Antiviral oligonucleotides

ABSTRACT

Random sequence oligonucleotides that have antiviral activity are described, along with their use as antiviral agents. In many cases, the oligonucleotides are greater than 40 nucleotides in length and include chemical modifications, such as modified internucleotidic linkages and modifications at the 2′-position of the ribose moieties. Also described are methods for the prophylaxis or treatment of a viral infection in a human or animal, and a method for the prophylaxis treatment of cancer caused by oncoviruses in a human or animal. The methods typically involve administering to a human or animal in need of such treatment, a pharmacologically acceptable, therapeutically effective amount of at least one oligonucleotide that act by a sequence complementary mode of action.

RELATED APPLICATIONS

This application is related to Ser. No. 10/969,812 which is acontinuation-in-part of Vaillant & Juteau U.S. application Ser. No.10/661,402, filed Sep. 12, 2003, Ser. Nos. 10/661,088, 10/661,099,10/661,403, 10/661,097 and 10,661/415, each of these are acontinuation-in-part of Vaillant & Juteau, PCT application Ser. No.PCT/IB03/04573, filed Sep. 11, 2003, entitled ANTIVIRALOLIGONUCLEOTIDES; said application Ser. Nos. 10/661,402, 10/661,088,10/661,099, 10/661,403, 10/661,097 and 10,661/415, also claim thebenefit of Vaillant & Juteau, U.S. Provisional Appl. 60/430,934, filedDec. 5, 2002 and of Vaillant & Juteau, U.S. Provisional Appl.60/410,264, filed Sep. 13, 2002, all of which are incorporated herein byreference in their entireties, including drawings.

FIELD OF THE INVENTION

The present invention relates to oligonucleotides having antiviralactivities and their use as therapeutic agents in viral infectionscaused by human and animal viruses and in cancers caused by oncogeneviruses and in other diseases whose etiology is viral-based.

BACKGROUND OF THE INVENTION

The following discussion is provided solely to assist the understandingof the reader, and does not constitute an admission that any of theinformation discussed or references cited constitute prior art to thepresent invention.

Many important infectious diseases afflicting mankind are caused byviruses. Many of these diseases, including rabies, smallpox,poliomyelitis, viral hemaoragghic fevers, hepatitis, yellow fever,immune deficiencies and various encephalitic diseases, are frequentlyfatal. Others are significant in that they are highly contagious andcreate acute discomfort such as influenza, measles, mumps andchickenpox, as well as respiratory or gastrointestinal disorders. Otherssuch as rubella and cytomegalovirus can cause congenital abnormalities.Finally there are viruses, known as oncoviruses, which can cause cancerin humans and animals.

Among viruses, the family of Herpesviridae is of great interest. TheHerpesviridae are a ubiquitous class of icoshedral, double stranded DNAviruses. Of over 100 characterized members of Herpesviridae (HHV), onlyeight infect humans. The best known among these are Herpes simplex type1 (HSV-1), Herpes simplex type 2 (HSV-2), Varicella zoster (chicken poxor shingles), cytomegalovirus (CMV) and Epstein-Barr virus (EBV). Theprevalence of Herpes viruses in humans is high, affecting at least onethird of the worldwide population; and in the United States, 70-80% ofthe population have some kind of Herpes infection. While the pathologyof Herpes infections are usually not dangerous, as in the case of HSV-1which usually only causes short lived lesions around the mouth and face,these viruses are also known to be the cause of more dangerous symptoms,which vary from genital ulcers and discharge to fetal infections whichcan lead to encephalitis (15% mortality) or disseminated infection (40%mortality).

Herpes viruses are highly disseminated in nature and highly pathogenicfor man. For example, Epstein-Barr virus (EBV) is known to causeinfectious mononucleosis in late childhood or adolescence or in youngadults. The hallmarks of acute infectious mononucleosis are sore throat,fever, headache, lymphadenopathy, enlarged tonsils and atypical,dividing lymphocytes in the peripheral blood. Other manifestationsfrequently include mild hepatitis, splenomegaly and encephalitis. EBV isalso associated with two forms of cancer: Burkitt's lymphoma (BL) andthe nasopharyngeal carcinoma (NPC). In endemic areas of equatorialAfrica, BL is the most common childhood malignancy, accounting forapproximately 80% of cancers in children. While moderately observed inNorth American Caucasians, NPC is one of the most common cancers inSouthern China with age incidence of 25 to 55 years. EBV, like thecytomegalovirus, is also associated with post-transplantlymphoproliferative disease, which is a potentially fatal complicationof chronic immunosuppression following solid organ or bone marrowtransplantation.

Other diseases are also associated with HSV, including skin and eyeinfections, for example, chorioretinitis or keratoconjunctivitis.Approximately 300,000 cases of HSV infections of the eye are diagnosedyearly in the United States.

AIDS (acquired immunodeficiency syndrome) is caused by the humanimmunodeficiency virus (HIV). By killing or damaging cells of the body'simmune system, HIV progressively destroys the body's ability to fightinfections and certain cancers. There are currently approximately 42million people living with HIV/AIDS worldwide. A total of 3.1 millionpeople died of HIV/AIDS related causes in 2002. The ultimate goal ofanti-HIV drug therapy is to prevent the virus from reproducing anddamaging the immune system. Although substantial progress has been madeover the past fifteen years in the fight against HIV, a cure stilleludes medical science. Today, physicians have more than a dozenantiretroviral agents in three different drug classes to manage thedisease. Typically, drugs from two or three classes are prescribed in avariety of combinations known as HAART (Highly Active AntiRetroviralTreatment). HAART therapies typically comprise two nucleoside reversetranscriptase inhibitors drugs with a third drug, either a proteaseinhibitor or a non-nucleoside reverse transcriptase inhibitor. Clinicalstudies have shown that HAART is the most effective means of reducingviral loads and minimizing the likelihood of drug resistance.

While HAART has been shown to reduce the amount of HIV in the body,commonly known as viral load, tens of thousands of patients encountersignificant problems with this therapy. Some side effects are seriousand include abnormal fat metabolism, kidney stones, and heart disease.Other side effects such as nausea, vomiting, and insomnia are lessserious, but still problematic for HIV patients that need chronic drugtherapy for a lifetime.

Currently approved anti-HIV drugs work by entering an HIV infected CD4+T cell and blocking the function of a viral enzyme, either the reversetranscriptase or a protease. HIV needs both of these enzymes in order toreproduce. However, HIV frequently mutates, rendering reversetranscriptase or protease inhibitor drugs ineffective against theseresistant strains. Once resistance occurs, viral loads increase anddictate the need to switch the ineffective agent for anotherantiretroviral agent. Unfortunately, when a virus becomes resistant toone drug in a class, other drugs in that class may also become lesseffective. This phenomenon, known as cross-resistance, occurs becausemany anti-HIV drugs work in a similar fashion. The occurrence of drugcross-resistance is highly undesirable because it reduces the availablenumber of treatment options for patients.

There is therefore a great need for the development of other antiviralagents effective against HIV that work through other mechanisms ofaction against which the virus has not developed resistance. This isbecoming especially important in view of recent data showing that 1 outof 10 patients newly diagnosed with HIV in Europe, is infected with astrain of HIV already resistant to at least one of the approved drugs onthe market.

Respiratory syncytial virus (RSV) causes upper and lower respiratorytract infections. It is a negative-sense, enveloped RNA virus and ishighly infectious. It commonly affects young children and is the mostcommon cause of lower respiratory tract illness in infants. RSVinfections are usually associated with moderate-to-severe cold-likesymptoms. However, severe lower respiratory tract disease may occur atany age, especially in elderly or immunocompromised patients. Childrenwith severe infections may require oxygen therapy and, in certain cases,mechanical ventilation. According to the American Medical Association,an increasing number of children are being hospitalized forbronchiolitis, often caused by RSV infection. RSV infections alsoaccount for approximately one-third of community-associated respiratoryvirus infections in patients in bone marrow transplant centers. In theelderly population, RSV infection has been recently recognized to bevery similar in severity to influenza virus infection.

Influenza (INF), also known as the flu, is a contagious disease that iscaused by the influenza virus. It attacks the respiratory tract inhumans (nose, throat, and lungs). An average of about 36,000 people peryear in the United States die from influenza, and 114,000 per yearrequire hospitalization as a result of influenza. Influenza has recentlybecome a more serious concern with the emergence of highly pathogenicstrains previously only found in animals (e.g. avian flu).

In all infectious diseases, the efficacy of a given therapy oftendepends on the host immune response. This is particularly true forherpes viruses, where the ability of all herpes viruses to establishlatent infections results in an extremely high incidence of reactivatedinfections in immunocompromised patients. In renal transplantrecipients, 40% to 70% reactivate latent HSV infections, and 80% to 100%reactivate CMV infections. Such viral reactivations have also beenobserved with AIDS patients.

The hepatitis B virus (HBV) is a DNA virus that belongs to theHepadnaviridae family of viruses. HBV causes hepatitis B in humans. Itis estimated that 2 billion people have been infected (1 out of 3people) in the world. About 350 million people remain chronicallyinfected and an estimated 1 million people die each year from hepatitisB and its complications. HBV can cause lifelong infection, cirrhosis ofthe liver, liver cancer, liver failure, and death. The virus istransmitted through blood and bodily fluids. This can occur throughdirect blood-to-blood contact, unprotected sex, use of unsterileneedles, and from an infected woman to her newborn during the deliveryprocess. Most healthy adults (90%) who are infected will recover anddevelop protective antibodies against future hepatitis B infections. Asmall number (5-10%) will be unable to get rid of the virus and willdevelop chronic infections while 90% of infants and up to 50% of youngchildren develop chronic infections when infected with the virus.Alpha-interferon is the most frequent type of treatment used.Significant side effects are related to this treatment includingflu-like symptoms, depression, rashes, other reactions and abnormalblood counts. Another treatment option includes 3TC which also has manyside effects associated with its use. In the last few years, there havebeen an increasing number of reports showing that patients treated with3TC are developing resistant strains of HBV. This is especiallyproblematic in the population of patients who are co-infected with HBVand HIV. There is clearly an urgent need to develop new antiviraltherapies against this virus.

Hepatitis C virus (HCV) infection is the most common chronic bloodborneinfection in the United States where the number of infected patientslikely exceeds 4 million. This common viral infection is a leading causeof cirrhosis and liver cancer, and is now the leading reason for livertransplantation in the United States. Recovery from infection isuncommon, and about 85 percent of infected patients become chroniccarriers of the virus and 10 to 20 percent develop cirrhosis. It isestimated that there are currently 170 million people worldwide who arechronic carriers. According to the Centers for Disease Control andPrevention, chronic hepatitis C causes between 8,000 and 10,000 deathsand leads to about 1,000 liver transplants in the United States aloneeach year. There is no vaccine available for hepatitis C. Prolongedtherapy with interferon alpha, or the combination of interferon withRibavirin, is effective in only about 40 percent of patients and causessignificant side effects.

Today, the therapeutic outlook for viral infections in general is notfavourable. In general, therapies for viruses have mediocre efficaciesand are associated with strong side effects which either prevent theadministration of an effective dosage or prevent long term treatment.Three clinical situations which exemplify these problems areherpesviridae, HIV and RSV infections.

In the case of herpesviridae, there are five major treatments currentlyapproved for use in the clinic: idoxuridine, vidarabine, acyclovir,foscarnet and ganciclovir. While having limited efficacy, thesetreatments are also fraught with side effects. Allergic reactions havebeen reported in 35% of patients treated with idoxuridine, vidarabinecan result in gastrointestional disturbances in 15% of patients andacyclovir, foscarnet and ganciclovir, being nucleoside analogs, affectDNA replication in host cells. In the case of ganciclovir, neutropeniaand thrombocytopenia are reported in 40% of AIDS patients treated withthis drug.

While there are many different drugs currently available for thetreatment of HIV infections, all of these are associated with sideeffects potent enough to require extensive supplemental medication togive patients a reasonable quality of life. The additional problem ofdrug resistant strains of HIV (a problem also found in herpesviridaeinfections) usually requires periodic changing of the treatment cocktailand in some cases, makes the infection extremely difficult to treat.

The treatment of RSV infections in young infants is another example ofthe urgent need for new drug development. In this case, the usual lineof treatment is to deliver Ribavirin by inhalation using asmall-particule aerosol in an isolation tent. Not only is Ribavirin onlymildly effective, but its use is associated with significant sideeffects. In addition, the potential release of the drug has caused greatconcern in hospital personnel because of the known teratogenicity ofRibavirin.

It is clear that for any new emerging antiviral drug being developed, itwould be highly desirable to incorporate the three following features:1—improved efficacy; 2—reduced risks of side effects and 3—a mechanismof action which is difficult for the virus to overcome by mutation.

Several attempts to inhibit particular viruses by various antisenseapproaches have been made.

Zamecnik et al. have used ONs specifically targeted to the reversetranscriptase primer site and to splice donor/acceptor sites (Zamecnik,et al (1986) Proc. Natl. Acad. Sci. USA 83:4143-) (Goodchild & Zamecnik(1989) U.S. Pat. No. 4,806,463).

Crooke and coworkers. (Crooke et al. (1992) Antimicrob. AgentsChemother. 36:527-532) described an antisense against HSV-1.

Draper et al. (1993) (U.S. Pat. No. 5,248,670) reported antisenseoligonucleotides having anti-HSV activity containing the Cat sequenceand hybridizing to the HSV-1 genes UL13, UL39 and UL40.

Kean et al. (Biochemistry (1995) 34:14617-14620) reported testing ofantisense methylphosphonate oligomers as anti-HSV agents.

Peyman et al. (Biol Chem Hoppe Seyler (1995) March; 376:195-198) havereported testing specific antisense oligonucleotides directed againstthe IE110 and the UL30 mRNA of HSV-1 for their antiviral properties.

Oligonucleotides or oligonucleotide analogs targeting CMV mRNAs codingfor IE1, IE2 or DNA polymerase were reported by Anderson et al (1997)(U.S. Pat. No. 5,591,720)

Hanecak et al (1999) (U.S. Pat. No. 5,952,490) have described modifiedoligonucleotides having a conserved G quartet sequence and a sufficientnumber of flanking nucleotides to significantly inhibit the activity ofa virus such as HSV-1.

Jairath et al (Antiviral Res. (1997) 33:201-213) have reported antisenseoligonucleotides against RSV.

Torrence et al (1999) (U.S. Pat. No. 5,998,602) have reported compoundscomprising an antisense component complementary to a single strandedportion of the RSV antigenomic strand (the mRNA strand), a linker and aoligonucleotide activator of RNase L.

Qi et al. (Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi (2000)14:253-256) have reported testing antisense PS-oligonucleotides (PS-ONs)in Coxsackie virus B3.

International publication WO9203051 (Roizman and Maxwell) describesmethylphosphonate antisense oligomers which are complementary to vitalregions of HSV viral genome or mRNA transcripts thereof which exhibitantiviral activity.

Guanosine/thymidine or guanosine-rich phosphorothioateoligodeoxynucleotides (GT-PS-ONs) have been reported to have antiviralactivity. The article stated that “several different PS-containingGT-rich ONs (B106-140, I100-12, and G106-57) all 26 or 27 nt in length,were just as effective at reducing HIV-2 titers as GT-rich ONsconsisting of 36 (B106-96, B106-97) or 45 nt (Table 4).” (Fennewald etal., Antiviral Res. (1995) 26:37-54).

In U.S. Pat. No. 6,184,369, anti-HIV, anti-HSV, and anti-CMVoligonucleotides containing a high percentage of guanosine bases aredescribed. In preferred embodiments, the oligonucleotide has a threedimensional structure and this structure is stabilized by guanosinetetrads. In a further embodiment, the oligonucleotide compositions ofthe invention have two or more runs of two contiguous deoxyguanosines.The patent claims a G-rich oligodeoxynucleotide (ODN) that includes atleast two G residues in at least two positions.

Cohen et al. (U.S. Pat. Nos. 5,264,423 and 5,276,019) described theinhibition of replication of HIV, and more particularly to PS-ODNanalogs that can be used to prevent replication of foreign nucleic acidsin the presence of normal living cells. Cohen et al describe antiviralactivity of antisense PS-ODNs specific to a viral sequence. They alsodescribe testing polyA, polyT and polyC PS-ODN sequences of 14, 18, 21and 28-mers and indicate an antiviral effect of those PS-ODNs.

Matsukura et al. (Matsukura et al (1987) Proc Natl Acad Sci USA84:7706-7710) later published the result described in Cohen et al, U.S.patents above.

Gao et al (Gao et al (1989) J Biol Chem 264 :11521-11526), describe theinhibition of replication of HSV-2, by PS-ODNs by testing of polyA,polyT and polyC PS-ODN sequences in sizes of 7, 15, 21 and 28nucleotides.

Archambault, Stein and Cohen (Archambault et al (1994) Arch Virol139:97109) report that a PS-ODN polyC of 28 nucleotides is not effectiveagainst HSV-1.

Stein et al (Stein et al. (1989) AIDS Res Hum Retrovir 5:639-646),published results concerning additional data on anti-HIV ODNs, generallyof 21-28 nucleotides in length.

Marshal et al. (Marshall et al. (1992) Proc. Natl. Acad. Sci. USA89:6265-6269) describe anti-HIV-1 effect of phosphorothioate andphosphorothioate poly-C oligos of 4-28 nucleotides in length.

Stein & Cheng (Stein et al. (1993) Science 261 :1004-1012), in a reviewarticle, mention the antiviral activity of non specific ODNs of 28nucleotides, stating that “the anti-HIV properties of PS oligos aresignificantly influenced by non-sequence-specific effects, that is, theinhibitory effect is independent of the base sequence.”

In a review article Lebedeva & Stein (Lebedeva et al (2001) Annul RevPharmacol 41:403-419) report a variety of non-specific protein bindingactivity of PS-ODNs, including viral proteins. They state that “thesemolecules are highly biologically active, and it is often relativelyeasy to mistake artifact for antisense”.

Rein et al. (U.S. Pat. No. 6,316,190) reported a GT rich ON decoy linkedto a fusion partner and binding to the HIV nucleocapsid, which can beused as an antiviral compound. Similarly, Campbell et al. (Campbell etal (1999) J. Virol. 73 :2270-2279) reported PO-ODN with a TGTGT motifbinding specifically to the nucleocapsid of HIV but with no referencesto an antiviral activity.

Feng at al. (Feng et al. (2002) J. Virol. 76 :11757-11762) describedA(n) and TG(n) PO-ODNs binding to the recombinant HIV nucleocapsid butwith no data nor references to an anti-HIV activity.

Antisense ODNs developed as anticancer agents, antiviral agents, or totreat others diseases are typically approximately 20 nucleotides inlength. In a review article (Stein, C A, (2001) J. Clin. Invest.108:641-644), it is affirmed that “the length of an antisenseoligonucleotide must be optimized: If the antisense oligonucleotide iseither too long or too short, an element of specificity is lost. At thepresent time, the optimal length for an antisense oligonucleotide seemsto be roughly 16-20 nucleotides”. Similarly, in another review article(Crooke, S T (2000) Methods Enzymol. 313:3-45) it is stated that“Compared to RNA and RNA duplex formation, a phosphorothioateoligodeoxynucleotide has a T_(m) approximately −2.2° lower per unit.This means that to be effective in vitro, phosphorothioateoligodeoxynucleotides must typically be 17- to −20-mer in length . . .”.

Caruthers and co-workers (Marshall et al. (1992) Proc. Natl. Acad. Sci.USA 89:6265-6269) reported anti-HIV activity of phosphorodithioate ODNs(PS2-ODNs) for a 12mer polycytidine-PS2-ODN and for a 14mer PS2-ODN. Noother sizes were tested for anti-HIV activity. They also reported theinhibition of HIV reverse transcriptase (RT) for 12, 14, 20 and 28merpolycytidine-PS2-ODNs. Later, this group (Marshal et al (1993) Science259:1564-1570) reported results showing sequence specific inhibition ofthe HIV RT. The same group published data for PS2-ODNs in severalpatents. In U.S. Pat Nos. 5,218,103 and 5,684.148, PS2-ODN structure andsynthesis is described. In U.S. Pat. Nos. 5,452,496, 5,278,302, and5,695,979 inhibition of HIV RT is described for PS2-ODNs not longer than15 bases. In U.S. Pat. Nos. 5,750,666 and 5,602,244, antisense activityof PS2-ODNs is described.

Oligonucleotides modified at the 2′ position of the ribose and theiruses in antisense strategies have been evaluated, e.g., as described inthe references cited below.

Inoue and coworkers (Inoue et al. (1985) Nucleic Acids Res. 16:165168)describe the synthesis and properties of oligos(2′-O-methylribonucleotides). The same group (Inoue et al. (1987) FEBSLetter 215:327-330) reported that no RNAse H mediated mRNA cleavageoccurs when the oligonucleotide contains all 2′-O-methylribonucleotides.With mixed oligonucleotides i.e. oligonucleotides having unmodified and2′-O-methylribonucleotides, they report sequence specific RNAse Hhydrolysis of the nucleic acid complex formed by RNA and2′-O-methylribonucleotides.

Fully 2′-O-methylated and phosphorothioated oligonucleotides which donot support RNase H-mediated cleavage of target mRNA, were used todetermine if active antisense oligonucleotides inhibited ICAM-1expression by an RNase H-dependent mechanism (Chiang et al., (1991) J.Biol. Chem. 266:18162-18171). They stated that these antisenseoligonucleotides may be useful as therapeutic agents.

Oligonucleotides with 2′-sugar modifications including 2′-O-methyl,2′-O-propyl, 2′-O-pentyl, and 2′-fluoro were analyzed for antisenseactivity. Evaluation of antisense activities of uniformly 2′-modifiedoligonucleotides revealed that these compounds were completelyineffective in inhibiting gene expression. Activity was restored if thecompound contained a stretch of at least five 2′-deoxyribonucleotideresidues. This minimum deoxyribonucleotide length correlated perfectlywith the minimum length required for efficient RNase H activation invitro. (Monia et al., 1993, J. Biol. Chem. 268:14514.)

Yu et al. ((1996) Bioorganic. Med. Chem. 4:1685-1692) reported thathybrid antisense oligonucleotides having phosphorothioate,phosphodiester, or mixed backbones with a portion of 2′-O-methylmodified sugars have a specific anti-HIV activity measured by p24 ELISAquantification.

It is reported that correct splicing was efficiently restored whenphosphorothioated 2′-O-methyl-oligoribonucleotides were targeted to theaberrant splice sites of IVS2-654 pre-mRNA expressed in mammalian cellsstably transformed with this mutated human beta-globin gene.(Sierakowska, et al (1996) Proc. Natl. Acad. Sci. USA 93:12840-12844.)

A review article, Agrawal ((1999) Biochim. Biophys. Acta 1489:53-68)suggests that for optimum activity, antisense oligonucleotides shouldhave a combination of various properties, instead of only increasedstability toward nucleases or high affnity to target RNA. Suchproperties include RNAse H activation. In a later review, Agrawal andKandimalla ((2000) Mol. Med. Today 6:72-81) say that mixed backboneoligonucleotides, including 2′-O-methyl modifications, have become thechoice for second-generation antisense oligonucleotides for theirimproved characteristics including RNAse H activation. An antisenseoligo should posses certain important characteristics such as theability to activate RNAse H upon binding to the target RNA. (Agrawal andKandimalla, 2001, Current Cancer Drug Target 1:197-209.) For mostantisense approaches target RNA cleavage by RNAse H is desired in orderto increase antisense potency. (Kurreck, 2003, Eur. J. Biochem.270:1628-1644.)

Many studies describe the use of the 2′-O-methoxyethyl modification inantisense oligonucleotides. An example is a study using a gapped 2′modified oligonucleotide antisense described in Zellweger et al. ((2001)J. Pharmacol. Experimental Therapeutics 298:934-940). Another exampleshows inhibition of the formation of the translation initiation complexusing RNase H independent 2′-O-methoxyethyl antisense. (Baker et al.1997) J. Biol. Chem. 272 :1994-12000.)

Kuwasaki et al. (2003) J. Antimicrob. Chemother. 51:813-819, describesthe design of a highly nuclease-resistant, dimeric hairpinguanosine-quadruplex containing 2′-O-methyl groups on the nucleosidesand sulphur groups on the internucleotidic bonds, and its anti-HIV-1activity in cultured cells.

Mou and Gray (2002) (Nucleic Acids Res. 30:749-758), indicates that,compared with typical phosphorothioate-DNA oligomers, the addition ofthe 2′-O-methyl modification lowers the non-specific protein bindingproperty. The protein binding affinities of g5p for a 36mersoligonucleotide increased in the order ofdA₃₆<rA₃₆<2′-O-MeA₃₆<S-rA₃₆<<S-2′-O-MeA₃₆<S-dA₃₆ (where d=deoxy, r=ribo,2′-O-Me=2′-O-methyl, S=phosphorothioate). This order was in agreementwith the order of S-RNA<<S-2′-O-MeRNA<S-DNA reported in Kandimalla etal. ((1998) Bioorganic Med Chem Lett. 8:2103-2108) for the non-specificbinding of plasma proteins, such as human serum albumin, γ-globulin andfibrinogen for these oligomer modifications.

U.S. Pat. Nos. 5,591,623 and 5,514,788 describe compositions and methodsfor the treatment and diagnosis of diseases amenable to treatmentthrough modulation of the synthesis or metabolism of intercellularadhesion molecules. In accordance with preferred embodiments,oligonucleotides are described which are specifically hybridizable withnucleic acids encoding intercellular adhesion genes. The inventiondescribes the synthesis of 2′-O-methyl phosphorothioate oligonucleotidesand their use as antisense.

U.S. Pat. Nos. 5,652,355, 6,143,881 and 6,346,614 describe hybridoligonucleotides (containing segments of deoxy- and ribo nucleotides)that resist nucleolytic degradation, form stable duplexes with RNA orDNA, and activate RNase H when hybridized with RNA. It is indicated thatone property of phosphorothioate 2′-O-methyl-oligonucleotide is thenon-activation of RNAse H. In one aspect, the invention provides hybridoligonucleotides that are effective in inhibiting viruses, pathogenicorganisms, or the expression of cellular genes. A feature ofoligonucleotides according to this aspect of the invention is thepresence of deoxyribonucleotides. Oligonucleotides according to theinvention contain at least one deoxyribonucleotide. The nucleotidesequence of oligonucleotides according to this aspect of the inventionis complementary to a nucleic acid sequence that is from a virus, apathogenic organism or a cellular gene.

U.S. Pat. Nos. 5,591,721 and 6,608,035 describe a method ofdown-regulating the expression of a gene in an animal by the oraladministration of an oligonucleotide whose nucleotide sequence iscomplementary to the targeted gene. Thus, because of the propertiesdescribed in the patent, such oligonucleotides are said to be usefultherapeutically by their ability to control or down-regulate theexpression of a particular gene in an animal. The hybrid DNA/RNAoligonucleotides useful in the method of the invention resistnucleolytic degradation, form stable duplexes with RNA or DNA, andpreferably activate RNase H when hybridized with RNA. Theoligonucleotides according to the invention are reported to be effectivein inhibiting the expression of various genes in viruses, pathogenicorganisms, or in inhibiting the expression of cellular genes. Thus,oligonucleotides according to the method of the invention have anucleotide sequence which is complementary to a nucleic acid sequencethat is from a virus, a pathogenic organism or a cellular gene.

U.S. Pat. No. 6,608,035 presents data indicating that a phosphorothioateoligonucleotide is not stable in the stomach after 6 hours but a hybridphosphorothioate oligonucleotide containing 2′-O-methyl ribonucleotideat the 3′ and 5′ ends and a deoxyribonucleotide interior is more stablein the stomach but partially degraded.

SUMMARY OF THE INVENTION

The present invention involves the discovery that oligonucleotides(ONs), e.g., oligodeoxynucleotides (ODNs), including highly modifiedoligonucleotides, can have a broadly applicable, sequence independentantiviral activity. Advantageous modifications include modifiedinternucleotidic linkages and 2′-modifications. It is not necessary forthe oligonucleotide to be complementary to any viral sequence or to havea particular distribution of nucleotides in order to have antiviralactivity. Such an oligonucleotide can even be prepared as a randomer,such that there will be at most a few copies of any particular sequencein a preparation, e.g., in a 15 micromol randomer preparation 32 or morenucleotides in length.

In addition, the inventors have discovered that different lengtholigonucleotides have varying antiviral effect. For example, presentresults indicate that the length of antiviral oligonucleotide thatproduces maximal antiviral effect when modified with phosphorothioateinternucleotidic linkages is typically in the range of 40-120nucleotides. In view of the present discoveries concerning antiviralproperties of oligonucleotides, this invention provides oligonucleotideantiviral agents that can have activity against numerous differentviruses, and can even be selected as broad-spectrum antiviral agents.Such antiviral agents are particularly advantageous in view of thelimited antiviral therapeutic options currently available.

Therefore, the ONs, e.g., ODNs, of the present invention are useful intherapy for treating or preventing viral infections or for treating orpreventing tumors or cancers induced by viruses, such as oncoviruses(e.g., retroviruses, papillomaviruses, and herpesviruses), and intreating or preventing other diseases whose etiology is viral-based.Such treatments are applicable to many types of patients and treatments,including, for example, the prophylaxis or treatment of viral infectionsin immunosuppressed human and animal patients.

A first aspect of the invention concerns antiviral oligonucleotides,e.g., purified oligonucleotides, where the antiviral occurs principallyby a sequence independent, e.g., non-sequence complementary, mode ofaction, and formulations containing such oligonucleotides.

Oligonucleotides useful in the present invention can be of variouslengths, e.g., at least 6, 10, 14, 15, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 35, 38, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160,or more nucleotides in length. Likewise, the oligonucleotide can be in arange, e.g., a range defined by taking any two of the preceding listedvalues as inclusive end points of the range, for example 10-20, 20-30,20-40, 30-40, 30-50, 40-50, 40-60, 40-80, 50-60, 50-70, 60-70, 70-80,60-120, and 80-120 nucleotides. In particular embodiments, a minimumlength or length range is combined with any other of the oligonucleotidespecifications listed herein for the present antiviral oligonucleotides.

The antiviral nucleotide can include various modifications, e.g.,stabilizing modifications, and thus can include at least onemodification in the phosphodiester linkage and/or on the sugar, and/oron the base. For example, the oligonucleotide can include one or morephosphorothioate linkages, phosphorodithioate linkages, and/ormethylphosphonate linkages. Different chemically compatible modifiedlinkages can be combined, e.g., modifications where the synthesisconditions are chemically compatible. While modified linkages areuseful, the oligonucleotides can include phosphodiester linkages, e.g.,include at least one phosphodiester linkage, or at least 5, 10, 20, 30%or more phosphodiester linkages. Additional useful modificationsinclude, without restriction, modifications at the 2′-position of thesugar, such as 2′-O-alkyl modifications such as 2′-O-methylmodifications, 2′-amino modifications, 2′-halo modifications such as2′-fluoro; acyclic nucleotide analogs. Other modifications are alsoknown in the art and can be used. In particular embodiments, theoligonucleotide has modified linkages throughout, e.g.,phosphorothioate; has a 3′- and/or 5′-cap; includes a terminal 3′-5′linkage; the oligonucleotide is or includes a concatemer consisting oftwo or more oligonucleotide sequences joined by a linker(s).

The present invention further provides an oligonucleotide, wherein saidoligonucleotide is linked or conjugated at one or more nucleotideresidues, to a molecule modifying the characteristics of theoligonucleotide to obtain one or more characteristics selected from thegroup consisting of higher stability, lower serum interaction, highercellular uptake, higher viral protein interaction, an improved abilityto be formulated for delivery, a detectable signal, higher antiviralactivity, better pharmacokinetic properties, specific tissuedistribution, lower toxicity.

In certain embodiments, the oligonucleotide includes at least 10, 20,30, 40, 50, 60, 70, 80, 90, 95, or 100% modified linkages, e.g.,phosphorothioate, phosphorodithioate, and/or methylphosphonate.

In certain embodiments, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or95%, or all of the nucleotides are modified at the 2′-position of theribose, e.g., 2′-OMe, 2′-F, 2′-amino.

In certain embodiments modified linkages are combined with2′-modifications in oligonucleotides, for example, at least 30% modifiedlinkages and at least 30% 2′-modifications; or respectively at least 40%and 40%, at least 50% and 50%, at least 60% and 60%, at least 70% and70%, at least 80% and 80%, at least 90% and 90%, 100% and 100%. Incertain embodiments, the oligonucleotide includes at least 30, 40, 50,60, 70, 80, 90, or 100% modified linkages and at least 30, 40, 50, 60,70, 80, 90, or 100% 2′-modifications where embodiments include eachcombination of listed modified linkage percentage and 2′-modificationpercentage (e.g., at least 50% modified linkage and at least 80%2′-modifications, and at least 80% modified linkages and 100%2′-modifications). In particular embodiments of each of the combinationspercentages described, the modified linkages are phosphorothioatelinkages; the modified linkages are phosphorodithioate linkages; the2′-modifications are 2′-OMe; the 2′-modifications are 2′-fluoro; the2′-modifications are a combination of 2′-OMe and 2′-fluoro; the modifiedlinkages are phosphorothioate linkages and the 2′-modifications are2′-OMe; the modified linkages are phosphorothioate linkages and the2′-modifications are 2′-fluoro; the modified linkages arephosphorodithioate linkages and the 2′-modifications are 2′-OMe; themodified linkages are phosphorodithioate linkages and the2′-modifications are 2′-fluoro; the modified linkages arephosphorodithioate linkages and the 2′-modifications are a combinationof 2′-OMe and 2′-fluoro. In certain embodiments of oligonucleotides asdescribed herein that combine a particular percentage of modifiedlinkages and a particular percentage of 2′-modifications, theoligonucleotide is at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 110, or 120 nucleotides in length, or is in a length range definedby taking any two of the specified lengths as inclusive endpoints of therange.

In certain embodiments, all but 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of theinternucleotidic linkages and/or 2′-positions of the ribose moiety aremodified, e.g., with linkages modified with phosphorothioate,phosphorodithioate, or methylphosphonate linkages and/or 2′-OMe, 2′-F,and/or 2′-amino modifications of the ribose moiety.

In some embodiments, the oligonucleotide includes at least 1, 2, 3, or 4ribonucleotides, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90%, oreven 100% ribonucleotides.

In particular embodiments, the oligonucleotide includes non-nucleotidegroups in the chain (i.e., form part of the chain backbone) and/or asside chain moieties, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or even more,or up to 5, 10, 20% or more of the chain moieties and/or side chainmoieties.

In certain embodiments, the oligonucleotide is free ofself-complementary sequences longer than 5, 8, 10, 15, 20, 25, 30nucleotides; the oligonucleotide is free of catalytic activity, e.g.,cleavage activity against RNA; the oligonucleotide does not induce anRNAi mechanism.

In particular embodiments, the oligonucleotide binds to one or moreviral proteins; the sequence of the oligonucleotide (or a portionthereof, e.g., at least 20, 30, 40, 50, 60, 70% or more) is derived froma viral genome; the activity of an oligonucleotide with a sequencederived from a viral genome is not superior to a randomeroligonucleotide or a random oligonucleotide of the same length; theoligonucleotide includes a portion complementary to a viral sequence anda portion not complementary to a viral sequence; the sequence of theoligonucleotide is derived from a viral packaging sequence or otherviral sequence involved in an aptameric interaction; unless otherwiseindicated, the sequence of the oligonucleotide includes A(x), C(x),G(x), T(x), U(x), I(x), AC(x), AG(x), AT(x), AU(x), CG(x), CT(x), CU(x),GT(x), GU(x), TU(x), AI(x), IC(x), IG(x), IT(x) IU(x) where x is 2, 3,4, 5, 6, . . . 60 . . . 120 (in particular embodiments theoligonucleotide is at least 15, 20, 25, 29, 30, 32, 34, 35, 36, 38, 40,45, 46, 50, 60, 70, 80, 90, 100, 110, 120, 140, or 160 nucleotides inlength or is in a range defined by taking any two of the listed valuesas inclusive endpoints, or the length of the specified repeat sequenceis at least a length or in a length range just specified); theoligonucleotide includes a combination of repeat sequences (e.g., repeatsequences as specified above), including, for example, each combinationof the above monomer and/or dimer repeats taken 2, 3, or 4 at a time;the oligonucleotide is single stranded (RNA or DNA); the oligonucleotideis double stranded (RNA or DNA); the oligonucleotide includes at leastone Gquartet or CpG portion; the oligonucleotide includes a portioncomplementary to a viral mRNA and is at least 29, 37, or 38 nucleotidesin length (or other length as specified above); the oligonucleotideincludes at least one non-Watson-Crick oligonucleotide and/or at leastone nucleotide that participates in non-Watson-Crick binding withanother nucleotide and/or at least one nucleotide that cannot form basepairs with other nucleotides; the oligonucleotide is a randomoligonucleotide, the oligonucleotide is a randomer or includes arandomer portion, e.g., a randomer portion that has a length of at least5, 10, 15, 20, 25, 30, 35, 40 or more contiguous oligonucleotides or alength as specified above for oligonucleotide length or at least 10, 20,30, 40, 50, 60, 70, 80, 90% or all the nucleotides are randomer; theoligonucleotide is linked or conjugated at one or more nucleotideresidues to a molecule that modifies the characteristics of theoligonucleotide, e.g. to provide higher stability (such as stability inserum or stability in a particular solution), lower serum interaction,higher cellular uptake, higher viral protein interaction, improvedability to be formulated for delivery, a detectable signal, improvedpharmacokinetic properties, specific tissue distribution, and/or lowertoxicity.

It was also discovered that phosphorothioated ONs containing only (or atleast primarily) pyrimidine nucleotides, including cytosine and/orthymidine and/or other pyrimidines are resistant to low pH andpolycytosine oligonucleotides showed increased resistance to a number ofnucleases, thereby providing two important characteristics for oraladministration of an antiviral ON. Thus, in certain embodiments, theoligonucleotide has at least 80, 90, or 95, or 100% modifiedinternucleotidic linkages (e.g., phosphorothioate or phosphorodithoiate)and the pyrimidine content is more than 50%, more than 60%, more than70%, more than 80%, more than 90%, or 100%, i.e.; is a pyrimidineoligonucleotide or the cytosine content is more than 50%, more than 60%,more than 70%, more than 80%, morethan 90% or 100% i.e. is apolycytosine oligonucleotide. In certain embodiments, the length is atleast 29, 30, 32, 34, 36, 38, 40, 45, 50, 60, 70, or 80 nucleotides, oris in a range of 20-28, 25-35, 29-40, 30-40, 35-45, 40-50, 45-55, 50-60,55-65, 60-70, 65-75, or 70-80, or is in a range defined by taking anytwo of the listed values as inclusive endpoints of the range. Inparticular embodiment, the oligonucleotide is at least 50, 60, 70, 80,or 90% cytosine; at least 50, 60, 70, 80, or 90% thymidine (and may havea total pyrimidine content as listed above). In particular embodiments,the oligonucleotide contains a listed percentage of either cytosine orthymidine, and the remainder of the pyrimidine nucleotides are the otherof cytosine and thymidine. Also in certain embodiments, theoligonucleotide includes at least 10, 12, 14, 16, 18, 20, 25, 30, 35,40, or more contiguous pyrimidine nucleotides, e.g., as C nucleotides, Tnucleotides, or CT dinucleotide pairs. In certain embodiments, thepyrimidine oligonucleotide consists only of pyrimidine nucleotides;includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-pyrimidinemoieities; includes 1-5, 6-10, 11-15, or at least 16 non-pyrimidinebackbone moieties; includes at least one, 1-20, 1-5, 6-10, 11-15, or16-20 non-nucleotide moieties; includes at least one, 1-20, 1-5, 6-10,11-15, or 16-20 purine nucleotides. Preferably, in embodiments in whichnon-nucleotide moieities are present, the linkages between such moietiesor between such moieties and nucleotides are at least 25, 35, 50, 70,90, or 100% as resistant to acidic conditions as PS linkages in a 40-merpolyC oligonucleotide as evaluated by gel electrophoresis underconditions appropriate for the size and chemistry of theoligonucleotide.

Oligonucleotides can also be used in combinations, e.g., as a mixture.Such combinations or mixtures can include, for example, at least 2, 3,4, 5, 10, 20, 50, 100, 1000, 10000, 100,000, 1,000,000, or moredifferent oligonucleotides, e.g., any combination of oligonucleotidesare described herein. Such combinations or mixtures can, for example, bedifferent sequences and/or different lengths and/or differentmodifications and/or different linked or conjugated molecules. Inparticular embodiments of such combinations or mixtures, a plurality ofoligonucleotides have a minimum length or are in a length range asspecified above for oligonucleotides. In particular embodiments of suchcombinations or mixtures, at least one, a plurality, or each of theoligonucleotides can have any of the other properties specified hereinfor individual antiviral oligonucleoties (which can also be in anyconsistent combination).

In certain embodiments, the sequence of the oligonucleotide is notperfectly complementary to any equal length portion of the genomesequence of the target virus, or has less than 95, 90, 80, 70, 60, or50% complementarity to any equal length portion of the genomic sequenceof the target virus, the oligonucleotide sequence does not consistessentially of polyA, polyC, polyG, polyT, Gquartet, or a TG-richsequence.

As used in connection with the present oligos, the term “TG-rich”indicates that the sequence of the antiviral oligonucleotide consists ofat least 50 percent T and G nucleotides, or if so specified, at least60, 70, 80, 90, or 95% T and G, or even 100%.

In a related aspect, the invention provides a mixture of antiviraloligonucleotides that includes at least two different antiviraloligonucleotides as described herein, e.g., at least 2, 3, 4, 5, 7, 10,50, 100, 1000, 10,000, 100,000, 1,000,000, or even more.

As used herein in connection with oligonucleotides or other materials,the term “antiviral” refers to an effect of the presence of theoligonucleotides or other material in inhibiting production of viralparticles, i.e., reducing the number of infectious viral particlesformed, in a system otherwise suitable for formation of infectious viralparticles for at least one virus. In certain embodiments of the presentinvention, the antiviral oligonucleotides will have antiviral activityagainst multiple different viruses.

The term “antiviral oligonucleotide formulation” refers to a preparationthat includes at least one antiviral oligonucleotide that is adapted foruse as an antiviral agent. The formulation includes the oligonucleotideor oligonucleotides, and can contain other materials that do notinterfere with use of the formulation as an antiviral agent in vivo.Such other materials can include without restriction diluents,excipients, carrier materials, and/or other antiviral materials.

As used herein, the term “pharmaceutical composition” refers to anantiviral oligonucleotide formulation that includes a physiologically orpharmaceutically acceptable carrier or excipient. Such compositions canalso include other components that do not make the compositionunsuitable for administration to a desired subject, e.g., a human.

In the context of the present invention, unless specifically limited theterm “oligonucleotide (ON)” means oligodeoxynucleotide (ODN) oroligodeoxyribonucleotide or oligoribonucleotide. Thus, “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) and/ordeoxyribonucleic acid (DNA) and/or analogs thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases. Examples of modifications thatcan be used are described herein. Oligonucleotides that include backboneand/or other modifications can also be referred to as oligonucleosides.

In the present context, the phrase “modified internucleotidic linkage”refers to a linkage between nucleotides or nucleotide analogs in anoligonucleotide that differs from the phosphodiester linkage generallyfound in naturally-occurring polynucleotides. Examples includephosphorothioate linkages, phosphorodithioate linkages, andmethylphosphonate linkages.

Specification of particular lengths for oligonucleotides, e.g., at least20 nucleotides in length, means that the oligonucleotide includes atleast 20 linked nucleotides. Unless clearly indicated to the contrary,the oligonucleotide may also include additional, non-nucleotidemoieties, which may form part of the backbone of the oligonucleotidechain. Unless otherwise indicated, when non-nucleotide moieities arepresent in the backbone, at least 10 of the linked nucleotides arecontiguous.

As used in connection with an antiviral formulation, pharmaceuticalcomposition, or other material, the phrase “adapted for use as anantiviral agent” indicates that the material exhibits an antiviraleffect and does not include any component or material that makes itunsuitable for use in inhibiting viral production in an in vivo system,e.g., for administering to a subject such as a human subject.

As used herein in connection with antiviral action of an antiviraloligonucleotide, “sequence independent mode of action” indicates thatthe particular biological activity (e.g., antiviral activity) is notdependent on a particular oligonucleotide sequence in theoligonucleotide. For example, the activity does not depend on sequencedependent hybridization such as with antisense activity, or a particularsequence resulting in a sequence dependent aptameric interaction.Similarly, the phrase “non-sequence complementary mode of action”indicates that the mechanism by which the material exhibits an antiviraleffect is not due to hybridization of complementary nucleic acidsequences, e.g., an antisense effect. Conversely, a “sequencecomplementary mode of action” means that the antiviral effect of amaterial involves hybridization of complementary nucleic acid sequencesor sequence specific aptameric interaction. Thus, indicating that theantiviral activity of a material is due to a sequence independent modeof action” or that the activity is “not primarily due to a sequencecomplementary mode of action” means that the the activity of theoligonucleotide satisfies at least one of the 4 tests provided herein(see Example, 10). In particular embodiments, the oligonucleotidesatisfies test 1, test 2, test 3, test 4, or test 5; the oligonucleotidesatisfies a combination of two of the tests, i.e., tests 1 & 2; tests 1& 3; tests 1 & 4, tests 1 & 5, tests 2 & 3, tests 2 & 4, test 2 & 5,tests 3 & 4, tests 3 & 5, or tests 4 & 5; the oligonucleotide satisfiesa combination of 3 of the tests, i.e., tests 1, 2, and 3, tests 1, 2,and 4, test 1, 2, & 5, tests 1, 3, and 4, tests 1, 3, & 5, tests 2, 3,and 4, tests 2, 3, & 5, tests 3, 4, & 5; the oligonucleotide satisifiesall of tests 1, 2, 3, and 4.

As used herein in connection with administration of an antiviralmaterial, the term “subject” refers to a living higher organism,including, for example, animals such as mammals, e.g., humans, non-humanprimates, bovines, porcines, ovines, equines, dogs, and cats; birds(Aves),; and plants, e.g., fruit trees.

A related aspect concerns an antiviral oligonucleotide randomer orrandomer formulation that contains at least one randomer, where theantiviral activity of the randomer occurs principally by a sequenceindependent, e.g., non-sequence complementary mode of action. Such arandomer formulation can, for example, include a mixture of randomers ofdifferent lengths, e.g., at least 2, 3, 5, 10, or more differentlengths, or other mixtures as described herein.

As used herein in connection with oligonucleotide sequences, the term“random” characterizes a sequence or an ON that is not complementary toa viral mRNA, and which is selected to not form hairpins and not to havepalindromic sequences contained therein. When the term “random” is usedin the context of antiviral activity of an oligonucleotide toward aparticular virus, it implies the absence of complementarity to a viralmRNA of that particular virus. The absence of complementarity may bebroader, e.g., for a plurality of viruses, for viruses from a particularviral family, or for infectious human viruses.

In the present application, the term “randomer” is intended to mean asingle stranded DNA having a wobble (N) at every position, such asNNNNNNNNNN. Each base is synthesized as a wobble such that this ONactually exists as a population of different randomly generatedsequences of substantially the same size. It is recognized thatpreparation of such a randomer will normally generate a distribution ofsizes around a particular length (primarily shorter lengths); unlessclearly indicated to the contrary, in the present context such apreparation is regarded as a randomer of the particular length.

The phrase “derived from a viral genome” indicates that a particularsequence has a nucleotide base sequence that has at least 70% identityto a viral genomic nucleotide sequence or its complement (e.g., is thesame as or complementary to such viral genomic sequence), or is acorresponding RNA sequence. In particular embodiments of the presentinvention, the term indicates that the sequence is at least 70%identical to a viral genomic sequence of the particular virus againstwhich the oligonucleotide is directed, or to its complementary sequence.In particular embodiments, the identity is at least 80, 90, 95, 98, 99,or 100%.

The invention also provides an antiviral pharmaceutical composition thatincludes a therapeutically effective amount of a pharmacologicallyacceptable, antiviral oligonucleotide or mixture of oligonucleotides asdescribed herein, e.g., at least 6 nucleotides in length or other lengthas listed herein, where the antiviral activity of the oligonucleotideoccurs principally by a sequence independent, e.g., non-sequencecomplementary, mode of action, and a pharmaceutically acceptablecarrier. In particular embodiments, the oligonucleotide or a combinationor mixture of oligonucleotides is as specified above for individualoligonucleotides or combinations or mixtures of oligonucleotides. Inparticular embodiments, the pharmaceutical compositions are approved foradministration to a human, or a non-human animal such as a non-humanprimate.

In particular embodiments, the pharmaceutical composition is adapted forthe treatment, control, or prevention of a disease with a viraletiology; adapted for treatment, control, or prevention of a priondisease; is adapted for delivery by intraocular administration, oralingestion, enteric administration, inhalation, cutaneous, subcutaneous,intramuscular, intraperitoneal, intrathecal, intratracheal, orintravenous injection, or topical administration.

In particular embodiments, the pharmaceutical composition can beformulated for delivery by a mode selected from the group consisting ofbut not restricted to oral ingestion, oral mucosal delivery, intranasaldrops or spray, intraocular injection, subconjonctival injection, eyedrops, ear drops, by inhalation, intratracheal injection or spray,intrabronchial injection or spray, intrapleural injection,intraperitoneal injection perfusion or irrigation, intrathecal injectionor perfusion, intracranial injection or perfusion, intramuscularinjection, intravenous injection or perfusion, intraarterial injectionor perfusion, intralymphatic injection or perfusion, subcutaneousinjection or perfusion, intradermal injection, topical skin application,by organ perfusion, by topical application during surgery, intratumoralinjection, topical application, gastric injection perfusion orirrigation, enteral injection or perfusion, colonic injection perfusionor irrigation, rectal injection perfusion or irrigation, by rectalsuppository or enema, by urethral suppository or injection, intravesicalinjection perfusion or irrigation, or intraarticular injection.

In particular embodiments, the composition includes a delivery system,e.g., targeted to specific cells or tissues; a liposomal formulation,another antiviral drug, e.g., a non-nucleotide antiviral polymer, anantisense molecule, an siRNA, or a small molecule drug.

In particular embodiments, the antiviral oligonucleotide,oligonucleotide preparation, oligonucleotide formulation, or antiviralpharmaceutical composition has an in vitro IC₅₀ for a target virus(e.g., any of particular viruses or viruses in a group of viruses asindicated herein) of 10, 5, 2, 1, 0.50, 0.20, 0.10, 0.09. 0.08, 0.07,0.75, 0.06, 0.05, 0.045, 0.04, 0.035, 0.03, 0.025, 0.02, 0.015, or 0.01μM or less.

In particular embodiments of formulations, pharmaceutical compositions,and methods for prophylaxis or treatment, the composition or formulationis adapted for treatment, control, or prevention of a disease with viraletiology; is adapted for the treatment, control or prevention of a priondisease; is adapted for delivery by a mode selected from the groupconsisting of intraocular, oral ingestion, enterally, inhalation, orcutaneous, subcutaneous, intramuscular, or intravenous injectiondelivery; further comprises a delivery system, which can include or beassociated with a molecule increasing affinity with specific cells;further comprises at least one other antiviral drug in combination;and/or further comprises an antiviral polymer in combination.

In particular embodiments, the pharmaceutical composition contains atleast one polypyrimidine oligonucleotide as described herein. In view ofthe resistance to low pH discovered for polypyrimidine oligonucleoides;in certain embodiments such a composition is adapted for delivery to anacidic in vivo site, e.g., oral delivery or vaginal delivery.

In particular embodiments of compositions and formulations for oraladministration containing such polypyrimidine oligonucleotides, thecomposition or formulation is prepared in the form of a powder,granules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, emulsion (e.g., microemulsion), capsule,gel capsule, sachet, tablet, or minitablet. In certain embodiments,thickeners, flavoring agents, diluents, emulsifiers, dispersing aids orbinders may be included. In some embodiments, the oral formulations arethose in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants and/orchelators, e.g. and without restriction, fatty acids and/or esters orsalts thereof (for example, arachidonic acid, undecanoic acid, oleicacid, lauric acid, caprylic acid, capric acid, myristic acid, palmiticacid, stearic acid, linoleic acid, linolenic acid, dicaprate,tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium), bile acids and/or salts thereof (for example,chenodeoxycholic acid (CDCA) and ursodeoxychenedeoxycholic acid (UDCA),cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid,glycholic acid, glycodeoxycholic acid, taurocholic acid,taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodiumglycodihydrofusidate). Some embodiments include a combination ofpenetration enhancers, for example, fatty acids/salts in combinationwith bile acids/salts such as the sodium salt of lauric acid, capricacid and UDCA. Further exemplary penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.

In particular embodiments in which the oligonucleotides of the inventionare prepared in granular form (including sprayed dried particles) orcomplexed to form micro or nanoparticles, a complexing agent(s) is usedthat is selected, without restriction, from poly-amino acids;polyimines; polyacrytates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses, and starches, or more specifically selected from chitosan,N-trimethytchitosan, poly-L-lysine, polyhistidine, polyorithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylatc), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG).

In particular embodiments, the composition is adapted for vaginaladministration. In such embodiments, the composition may be prepared,without limitation, in the form of tablets, a solution, a cream, a gel,a suppository.

In particular embodiments, the composition is adapted for topicaladministration.

As used herein, the terms “polypyrimidine oligonucleotide” or“pyrimidine oligonucleotide” means an oligonucleotide that containsgreater than 50% pyrimidine nucleotides.

As used in relation to in vivo administration of the presentoligonucleotides and compositions, the term “acidic site” means a sitethat has a pH of less than 7. Examples include the stomach (pH generally1-2), the vagina (pH generally 4-5 but may be lower), and to a lesserdegree, the skin (pH generally 4-6).

As used herein, the phrase “adapted for oral delivery” and like termsindicate that the composition is sufficiently resistant to acidic pH toallow oral administration without a clinically excessive loss ofactivity, e.g., an excessive first pass loss due to stomach acidity ofless than 50% (or is indicated, less than 40%, 30%, 20%, 10%, or 5%).

As used herein, the phrase “adapted for vaginal administration” and liketerms indicate that the composition is prepared such that whenappropriately administered, the composition will not degrade to aclinically unacceptable extent (e.g., less than 50%, 40%, 30%, 20%, 10%,or 5% over a specified time for retention) and will remain substantiallyin the vagina (excluding material that is absorbed) for at least 1 hour(or if indicated, for at least 2 hr, 4 hr, 8 hr, 12 hr, 1 day, or 2days). Such retention may be due to any of a number of different factorsor combinations of factors, for example, due to physical form oradhesive properties, and the like.

As used herein in connection with antiviral oligonucleotides andformulations, and the like, in reference to a particular virus or groupof viruses the term “targeted” indicates that the oligonucleotide isselected to inhibit that virus or group of viruses. As used inconnection with a particular tissue or cell type, the term indicatesthat the oligonucleotide, formulation, or delivery system is selectedsuch that the oligonucleotide is preferentially present and/orpreferentially exhibits an antiviral effect in or proximal to theparticular tissue or cell type.

As used herein, the term “delivery system” refers to a component orcomponents that, when combined with an oligonucleotide (e.g., anantisense oligo, siRNA, or oligonucleotide as described herein),increases the amount of the oligonucleotide that contacts the intendedlocation in vivo, and/or extends the duration of its presence at thetarget, e.g., by at least 20, 50, or 100%, or even more as compared tothe amount and/or duration in the absence of the delivery system, and/orprevents or reduces interactions that cause side effects.

As used herein in connection with antiviral agents and other drugs ortest compounds, the term “small molecule” means that the molecularweight of the molecule is 1500 daltons or less. In some cases, themolecular weight is 1000, 800, 600, 500, or 400 daltons or less.

In another aspect, the invention provides a kit that includes at leastone antiviral oligonucleotide, antiviral oligonucleotide mixture,antiviral oligonucleotide formulation, or antiviral pharmaceuticalcomposition that includes such oligonucleotide, oligonucleotide mixture,or oligonucleotide formulation in a labeled package, where the antiviralactivity of the oligonucleotide occurs principally by a sequenceindependent e.g., non-sequence complementary, mode of action and thelabel on the package indicates that the antiviral oligonucleotide can beused against at least one virus.

In particular embodiments the kit includes a pharmaceutical compositionthat includes at least one antiviral oligonucletide as described herein.In one embodiment, the kit contains a mixture of at least two differentantiviral oligonucleotides. In one embodiment, the antiviraloligonucleotide is adapted for in vivo use in an animal and/or the labelindicates that the oligonucleotide or composition is acceptable and/orapproved for use in an animal; the animal is a mammal, such as human, ora non-human mammal such as bovine, porcine, a ruminant, ovine, orequine; the animal is a non-human animal; the animal is a bird, the kitis approved by a regulatory agency such as the U.S. Food and DrugAdministration or equivalent agency for use in an animal, e.g., a human.

In another aspect, the invention provides a method for selecting anantiviral oligonucleotide, e.g, a non-sequence complementary antiviraloligonucleotide, for use as an antiviral agent. The method involvessynthesizing a plurality of different random oligonucleotides, testingthe oligonucleotides for activity in inhibiting the ability of a virusto produce infectious virions, and selecting an oligonucleotide having apharmaceutically acceptable level of activity for use as an antiviralagent.

In particular embodiments, the different random oligonucleotidescomprises randomers of different lengths; the random oligonucleotidescan have different sequences or can have sequence in common, such as thesequence of the shortest oligos of the plurality; and/or the differentrandom oligonucleotides comprise a plurality of oligonucleotidescomprising a randomer segment at least 5 nucleotides in length or thedifferent random oligonucleotides include a plurality of randomers ofdifferent lengths. Other oligonucleotides, e.g., as described herein forantiviral oligonucleotides, can be tested in a particular system.

In yet another aspect, the invention provides a method for theprophylaxis or treatment of a viral infection in a subject byadministering to a subject in need of such treatment a therapeuticallyeffective amount of at least one pharmacologically acceptableoligonucleotide as described herein, e.g., a non-sequence complementaryoligonucleotide at least 6 nucleotides in length, or an antiviralpharmaceutical composition or formulation or mixture containing sucholigonucleotide(s). In particular embodiments, the virus can be any ofthose listed herein as suitable for inhibition using the presentinvention; the infection is related to a disease or condition indicatedherein as related to a viral infection; the subject is a type of subjectas indicated herein, e.g., human, non-human animal, non-human mammal,bird, plant, and the like; the treatment is for a viral disease ordisease with a viral etiology, e.g., a disease as indicated in theBackground section herein.

In yet another aspect, the invention provides a method for theprophylaxis or treatment of a viral infection in an acidic environnementin a subject, comprising administering to a subject in need of such atreatment a therapeutically effective amount of at least onepharmacologically acceptable antiviral pharmaceutical composition of theinvention, said composition being adapted for administration to anacidic in vivo site.

In particular embodiments, an antiviral oligonucleotide (oroligonucleotide formulation or pharmaceutical composition) as describedherein is administered; administration is a method as described herein;a delivery system or method as described herein is used; the viralinfection is of a DNA virus or an RNA virus; the virus is aparvoviridae, papovaviridae, adenoviridae, herpesviridae, poxviridae,hepadnaviridae, or papillomaviridae; the virus is a arenaviridae,bunyaviridae, calciviridae, coronaviridae, filoviridae, flaviridae,orthomyxoviridae, paramyxoviridae, picornaviridae, reoviridae,rhabdoviridae, retroviridae, or togaviridae; the herpesviridae virus isEBV, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, or HHV-8; the virus is HIV-1or HIV-2; the virus is respiratory syncytical virus (RSV); the virus isparainfluenza-3 virus; the virus is an influenza virus, e.g., influenzaA; the virus is HBV; the virus is smallpox virus or vaccinia virus; thevirus is a coronavirus; the virus is SARS virus; the virus is West NileVirus; the virus is a hantavirus; the virus is a parainfluenza virus;the virus is coxsackievirus; the virus is rhinovirus; the virus isyellow fever virus; the virus is dengue virus; the virus is hepatitis Cvirus; the virus is Ebola virus; the virus is Marburg virus; the virusis Lassa fever virus; the virus is Varicella Zoster Virus; the virus isEpstein Barr Virus; the virus is Human Herpesvirus 6A or 6B; the virusis HBV; the virus is parainfluenza virus; the virus is humanmetapneumovirus; the virus is Rift Valley fever virus; the virus isCrimean Congo Hemorrhagic Fever virus; the virus is Western EquineEncephalitis virus.

In particular embodiments, the oligonucleotide is a polypyrimidineoligonucleotide (or a formulation or pharmaceutical compositioncontaining such polypyrimidine oligonucleotide), which may be adaptedfor oral or vaginal administration, e.g., as described herein.

Similarly, in a related aspect, the invention provides a method for theprophylactic treatment of cancer caused by oncoviruses in a human oranimal by administering to a human or animal in need of such treatment,a pharmacologically acceptable, therapeutically effective amount of atleast one random oligonucleotide of at least 6 nucleotides in length (oranother length as described herein), or a formulation or pharmaceuticalcomposition containing such oligonucleotide. In one embodiment, amixture of oligonucleotides of the invention.

In particular embodiments, the oligonucleotide(s) is as described hereinfor the present invention, e.g., having a length as described herein; amethod of administration as described herein is used; a delivery systemas described herein is used.

The term “therapeutically effective amount” refers to an amount that issufficient to effect a therapeutically or prophylactically significantreduction in production of infectious virus particles when administeredto a typical subject of the intended type. In aspects involvingadministration of an antiviral oligonucleotide to a subject, typicallythe oligonucleotide, formulation, or composition should be administeredin a therapeutically effective amount.

In certain embodiments involving oligonucleotide formulations,pharmaceutical compositions, and/or treatment and prophylactic methodsdescribed herein, the oligonucleotide(s) having a sequence independentmode of action is not associated with a transfection agent; theoligonucleotide(s) having a sequence independent mode of action is notencapsulated in liposomes and/or non-liposomal lipid particles. Incertain embodiments, the oligonucleotide(s) having a sequenceindependent mode of action is in a pharmaceutical composition or isadministered in conjunction with (concurrently or sequentially) anantiviral oligonucleotide that acts principally by a sequence dependentmode of action, e.g., antisense oligonucleotide or siRNA, where theoligonucleotide(s) having a sequence dependent mode of action can beassociated with a transfection agent and/or encapsulated in liposomesand/or non-liposomal lipid particles.

In another aspect, the discovery that sequence independent, e.g.,non-sequence complementary, interactions produce effective antiviralactivity provides a method of screening to identify a compound thatalters binding of an oligonucleotide to a viral component, such as oneor more viral proteins (e.g., extracted or purified from a viral cultureof infected host organisms, or produced by recombinant methods). Forexample, the method can involve determining whether a test compoundreduces the binding of oligonucleotide to one or more viral components.

As used herein, the term “screening” refers to assaying a plurality ofcompounds to determine if they possess a desired property. The pluralityof compounds can, for example, be at least 10, 100, 1000, 10,000 or moretest compounds.

In particular embodiments, any of a variety of assay formats anddetection methods can be used to identify such alteration in binding,e.g., by contacting the oligonucleotide with the viral component(s) inthe presence and absence of a compound(s) to be screened (e.g., inseparate reactions) and determining whether a difference occurs inbinding of the oligo the viral component(s) in the presence of thecompound compared to the absence of the compound. The presence of such adifference is indicative that the compound alters the binding of therandom oligonucleotide to the viral component. Alternatively, acompetitive displacement can be used, such that oligonucleotide is boundto the viral component and displacement by added test compound isdetermined, or conversely test compound is bound and displacement byadded oligonucleotide is determined.

In particular embodiments, the oligonucleotide is as described hereinfor antiviral oligonucleotides; the oligonucleotide is at least 6, 8,10, 15, 20, 25, 29, 30, 32, 34, 36, 38, 40, 46, 50, 60, 70, 80, 90, 100,110, or 120 nucleotides in length or at least another length specifiedherein for the antiviral oligonucleotides, or is in a range defined bytaking any two of the preceding values as inclusive endpoints of therange; the test compound(s) is a small molecule; the test compound has amolecular weight of less than 400, 500, 600, 800, 1000, 1500, 2000,2500, or 3000 daltons, or is in a range defined by taking any two of thepreceding values as inclusive endpoints of the range; the viral extractor component is from a virus as listed herein; at least 100, 1000,10,000, 20,000, 50,000, or 100,000 compounds are screened; theoligonucleotide has an in vitro IC₅₀ of equal to or less than 10, 5, 2,1, 0.500, 0.200, 0.100, 0.075, 0.05, 0.045, 0.04, 0.035, 0.03, 0.025,0.02, 0.015, or 0.01 μM.

The present invention further provides oligonucleotides described inTable 21.

The present invention further provides an antiviral oligonucleotide asset forth in any one of REP 1001, REP 2001, REP 3007, REP 2004, REP2005, REP 2006, REP 2007, REP 2008, REP 2017, REP 2018, REP 2020, REP2021, REP 2024, REP 2036, A20, G20, C20, REP 2029, REP 2031, REP 2030,REP 2033, REP 2055, REP 2056, REP 2057, REP 2060 and REP 2107.

As used herein, the term “viral component” refers to a product encodedby a virus or produced by infected host cells as a consequence of theviral infection. Such components can include proteins as well as otherbiomolecules. Such viral components, can, for example, be obtained fromviral cultures, infected host organisms, e.g., animals and plants, orcan be produced from viral sequences in recombinant systems (prokaryotesand eukaryotes), as well synthetic proteins having amino acid sequencescorresponding to viral encoded proteins. The term “viral cultureextract” refers to an extract from cells infected by a virus that willinclude virus-specific products. Similarly, a “viral protein” refers toa virus-specific protein, usually encoded by a virus, but can also beencoded at least in part by host sequences as a consequence of the viralinfection.

In a related aspect, the invention provides an antiviral compoundidentified by the preceding method, e.g., a novel antiviral compound.

In a further aspect, the invention provides a method for purifyingoligonucleotides binding to at least one viral component from a pool ofoligonucleotides by contacting the pool with at least one viralcomponent, e.g., bound to a stationary phase medium, and collectingoligonucleotides that bind to the viral component(s). Generally, thecollecting involves displacing the oligonucleotides from the viralcomponent(s). The method can also involve sequencing and/or testingantiviral activity of collected oligonucleotides (i.e., oligonucleotidesthat bound to viral protein).

In particular embodiments, the bound oligonucleotides of the pool aredisplaced from the stationary phase medium by any appropriate method,e.g., using an ionic displacer, and displaced oligonucleotides arecollected. Typically for the various methods of displacement, thedisplacement can be performed in increasing stringent manner (e.g., withan increasing concentration of displacing agent, such as a saltconcentration, so that there is a stepped or continuous gradient), suchthat oligonucleotides are displaced generally in order of increasedbinding affinity. In many cases, a low stringency wash will be performedto remove weakly bound oligonucleotides, and one or more fractions willbe collected containing displaced, tighter binding oligonucleotides. Insome cases, it will be desired to select fractions that contain verytightly binding oligonucleotides (e.g., oligonucleotides in fractionsresulting from displacement by the more stringent displacementconditions) for further use.

Similarly, the invention provides a method for enrichingoligonucleotides from a pool of oligonucleotides binding to at least oneviral component, by contacting the pool with one or more viral proteins,and amplifying oligonucleotides bound to the viral proteins to providean enriched oligonucleotide pool. The contacting and amplifying can beperformed in multiple rounds, e.g., at least 1, 2, 3, 4, 5, 10, or moreadditional times using the enriched oligonucleotide pool from thepreceding round as the pool of oligonucleotides for the next round. Themethod can also involve sequencing and testing antiviral activity ofoligonucleotides in the enriched oligonucleotide pool following one ormore rounds of contacting and amplifying.

The method can involve displacing oligonucleotides from the viralcomponent (e.g., viral protein bound to a solid phase medium) with anyof a variety of techniques, such as those described above, e.g., using adisplacement agent. As indicated above, it can be advantageous to selectthe tighter binding oligonucleotides for further use, e.g., in furtherrounds of binding and amplifying. The method can further involveselecting one or more enriched oligonucleotides, e.g., high affinityoligonucleotides, for further use. In particular embodiments, theselection can include eliminating oligonucleotides that have sequencescomplementary to host genomic sequences (e.g., human) for a particularvirus of interest. Such elimination can involve comparing theoligonucleotide sequence(s) with sequences from the particular host in asequence database(s), e.g., using a sequence alignment program (e.g., aBLAST search), and eliminating those oligonucleotides that havesequences identical or with a particular level of identity to a hostsequence. Eliminating such host complementary sequences and/or selectingone or more oligonucleotides that are not complementary to hostsequences can also be done for the other aspects of the presentinvention.

In the preceding methods for identifying, purifying, or enrichingoligonucleotides, the oligonucleotides can be of types as describedherein. The above methods are advantageous for identifying, purifying orenriching high affinity oligonucleotides, e.g., from an oligonucleotiderandomer preparation.

In a related aspect, the invention concerns an antiviral oligonucleotidepreparation that includes one or more oligonucleotides identified usinga method of any of the preceding methods for identifying, obtaining, orpurifying antiviral oligonucleotides from an initial oligonucleotidepool, where the oligonucleotides in the oligonucleotide preparationexhibit higher mean binding affinity with one or more viral proteinsthan the mean binding affinity of oligonucletides in the initialoligonucleotide pool.

In particular embodiments, the mean binding affinity of theoligonucleotides is at least two-fold, 3-fold, 5-fold, 10-fold, 20-fold,50-fold, or 100-fold greater than the mean binding affinity ofoligonucleotides in the initial oligonucleotide pool, or even more; themedian of binding affinity is at least two-fold, 3-fold, 5-fold,10-fold, 20-fold, 50-fold, or 100-fold greater relative to the median ofthe binding affinity of the initial oligo pool, where median refers tothe middle value.

In yet another aspect, the invention provides an antiviral polymer mixthat includes at least one antiviral oligonucleotide and at least onenon-nucleotide antiviral polymer. In particular embodiments, theoligonucleotide is as described herein for antiviral oligonucleotidesand/or the antiviral polymer is as described herein or otherwise knownin the art or subsequently identified.

In yet another aspect, the invention provides an oligonucleotiderandomer, where the randomer is at least 6 nucleotides in length. Inparticular embodiments the randomer has a length as specified above forantiviral oligonucleotides; the randomer includes at least onephosphorothioate linkage, the randomer includes at least onephosphorodithioate linkage or other modification as listed herein; therandomer oligonucleotides include at least one non-randomer segment(such as a segment complementary to a selected virus nucleic acidsequence), which can have a length as specified above foroligonucleotides; the randomer is in a preparation or pool ofpreparations containing at least 5, 10, 15, 20, 50, 100, 200, 500, or700 micromol, 1, 5, 7, 10, 20, 50, 100, 200, 500, or 700 mmol, or 1 moleof randomer, or a range defined by taking any two different values fromthe preceding as inclusive end points, or is synthesized at one of thelisted scales or scale ranges.

Likewise, the invention provides a method for preparing antiviralrandomers, by synthesizing at least one randomer, e.g., a randomer asdescribed above.

As indicated above, for any aspect involving a viral infection or riskof viral infection or targeting to a particular virus, in particularembodiments the virus is as listed above.

The expression “human and animal viruses” is intended to include,without limitation, DNA and RNA viruses in general. DNA viruses include,for example, parvoviridae, papovaviridae, adenoviridae, herpesviridae,poxviridae, hepadnaviridae, and papillomaviridae. RNA viruses include,for example, arenaviridae, bunyaviridae, calciviridae, coronaviridae,filoviridae, flaviridae, orthomyxoviridae, paramyxoviridae,picornaviridae, reoviridae, rhabdoviridae, retroviridae, or togaviridae.

In connection with modifying characteristics of an oligonucleotide bylinking or conjugating with another molecule or moiety, themodifications in the characteristics are evaluated relative to the sameoligonucleotide without the linked or conjugated molecule or moiety.

Additional embodiments will be apparent from the Detailed Descriptionand from the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is concerned with the identification and use ofantiviral oligonucleotides that act by a sequence independent mechanism,and includes the discovery that for many viruses, the antiviral activityis greater for larger oligonucleotides, and is typically optimal foroligonucleotides that are 40 nucleotides or more in length.

In accordance with the present invention there is provided anoligonucleotide comprising at least one modified internucleotidiclinkage, wherein said oligonucleotide has an antiviral activity againsta target virus wherein said activity operates predominantly by asequence independent mode of action.

In accordance with the present invention, there is provided anoligonucleotide, having at least 50% of its nucleotides in saidoligonucleotide modified at the 2′-position of the ribose moiety andhaving at least 50% of its internucleotidic linkages modified, whereinsaid oligonucleotide has an antiviral activity against a target virus,said activity operating predominantly by a sequence independent mode ofaction. In one embodiment, 50%, 80% respectively. In one embodiment,80%, 80% respectively. In one embodiment, 90%, 90% respectively. In oneembodiment, 100%, 100% respectively.

Lengths & Not Self-Complementary

The present invention further provides an oligonucleotide having atleast 15 nucleotides in length. In one embodiment, at least 20nucleotides in length. In one embodiment, at least 25 nucleotides inlength. In one embodiment, at least 30 nucleotides in length. In oneembodiment, at least 35 nucleotides in length. In one embodiment, atleast 40 nucleotides in length. In one embodiment, at least 45nucleotides in length. In one embodiment, at least 50 nucleotides inlength. In one embodiment, at least 60 nucleotides in length. In oneembodiment, at least 80 nucleotides in length.

The present invention further provides an oligonucleotide having 20-30nucleotides in length. In one embodiment, 30-40 nucleotides in length.inone embodiment, 40-50 nucleotides in length. In one embodiment, 50-60nucleotides in length. In one embodiment, 60-70 nucleotides in length.In one embodiment, 70-80 nucleotides in length.

The present invention further provides an oligonucleotide which is freefrom self-complementary sequences of greater than 5 contiguousnucleotides. In one embodiment, greater than 10 contiguous nucleotides.In one embodiment, greater than 20 contiguous nucleotides.

The present invention further provides an oligonucleotide which is freeof catalytic activity.

Random

The present invention further provides an oligonucleotide having anantiviral activity against a target virus, and the sequence of saidoligonucleotide not being complementary to any equal length portion ofthe genomic sequence of said target virus.

The present invention further provides an oligonucleotide, wherein saidoligonucleotide is not complementary to any equal length portion of thegenomic sequence of a human pathogenic virus.

The present invention further provides an oligonucleotide, wherein saidoligonucleotide is not complementary to any equal length portion of thegenomic sequence of a human pathogenic virus sequenced as of Jan. 1st,2005.

The present invention further provides an oligonucleotide which is notcomplementary to any equal length portion of the genomic sequence of ahuman.

The present invention further provides an oligonucleotide which is notcomplementary to any equal length portion of the genomic sequence of oneor more animals selected from the group consisting of cattle, horse,swine, sheep, bird, dog, cat and fish.

RNA and Other Chain Moiety Limitations

The present invention further provides an oligonucleotide wherein atleast 30% of the nucleotides are ribonucleotides. In one embodiment, atleast 50% of the nucleotides are ribonucleotides. In one embodiment, atleast 70% of the nucleotides are ribonucleotides. In one embodiment, atleast 80% of the nucleotides are ribonucleotides. In one embodiment, atleast 90% of the nucleotides are ribonucleotides. In one embodiment, allof the nucleotides are ribonucleotides.

The present invention further provides an oligonucleotide comprising 1-4non-nucleotide chain moieties.

Randomer

The present invention further provides an oligonucleotide comprising atleast 10 contiguous nucleotides of randomer sequence. In one embodiment,at least 20 nucleotides of randomer sequence. In one embodiment, atleast 30 nucleotides of randomer sequence. In one embodiment, at least40 nucleotides of randomer sequence.

The present invention further provides an oligonucleotide wherein saidoligonucleotide is randomer oligonucleotide.

Homopolymer

The present invention further provides an oligonucleotide comprising ahomopolymer sequence of at least 10 contiguous A nucleotides. In oneembodiment, at least 10 contiguous T nucleotides In one embodiment, atleast 10 contiguous U nucleotides. In one embodiment, at least 10contiguous C nucleotides. In one embodiment, at least 10 contiguous Gnucleotides. In one embodiment, at least 10 contiguous I nucleotideanalogs.

Heterodimers

The present invention further provides an oligonucleotide comprising apolyAT sequence at least 10 nucleotides in length. In one embodiment, apolyAC sequence at least 10 nucleotides in length. In one embodiment, apolyAG sequence at least 10 nucleotides in length. In one embodiment, apolyAU sequence at least 10 nucleotides in length. In one embodiment, apolyAI sequence at least 10 nucleotides in length. In one embodiment, apolyGC sequence at least 10 nucleotides in length. In one embodiment, apolyGT sequence at least 10 nucleotides in length. In one embodiment, apolyGU sequence at least 10 nucleotides in length. In one embodiment, apolyGI sequence at least 10 nucleotides in length. In one embodiment, apolyCT sequence at least 10 nucleotides in length. In one embodiment, apolyCU sequence at least 10 nucleotides in length. In one embodiment, apolyCI sequence at least 10 nucleotides in length. In one embodiment, apolyTI sequence at least 10 nucleotides in length.

Modified Linkages, Including ps and ps2

The present invention further provides an oligonucleotide, wherein themodified linkages are selected from the group consisting ofphosphorothioate linkages, phosphorodithioate linkages, andboranophosphate linkages.

The present invention further provides an oligonucleotide wherein atleast 50% of the internucleotidic linkages are modified linkages. In oneembodiment, wherein at least 80% of the internucleotidic linkages aremodified linkages. In one embodiment, wherein at least 90% of theinternucleotidic linkages are modified linkages. In one embodiment,wherein all of the internucleotidic linkages are modified linkages.

The present invention further provides an oligonucleotide, wherein atleast 50% of the internucleotidic linkages are phosphorothioatelinkages. In one embodiment, wherein at least 80% of theinternucleotidic linkages are phosphorothioate linkages. In oneembodiment, wherein at least 90% of the internucleotidic linkages arephosphorothioate linkages. In one embodiment, wherein all of theinternucleotidic linkages are phosphorothioate linkages.

The present invention further provides an oligonucleotide, wherein atleast 50% of the internucleotidic linkages are phosphorodithioatelinkages. In one embodiment, wherein at least 80% of theinternucleotidic linkages are phosphorodithioate linkages. In oneembodiment, wherein all of the internucleotidic linkages arephosphorodithioate linkages.

2′-Modifications, Combinations with Modified Linkages

The present invention further provides an oligonucleotide, wherein saidoligonucleotide comprises at least one phosphodiester linkage. In oneembodiment, wherein said oligonucleotide comprises at least 10%phosphodiester linkages. In one embodiment, wherein said oligonucleotidecomprises at least 20% phosphodiester linkages.

In one embodiment, wherein at least 50% of the nucleotides in saidoligonucleotide are modified at the 2′-position of the ribose moiety. Inone embodiment, wherein at least 60% of the nucleotides in saidoligonucleotide are modified at the 2′-position of the ribose moiety. Inone embodiment, wherein at least 70% of the nucleotides in saidoligonucleotide are modified at the 2′-position of the ribose moiety. Inone embodiment, wherein at least 80% of the nucleotides in saidoligonucleotide are modified at the 2′-position of the ribose moiety. Inone embodiment, wherein at least 90% of the nucleotides in saidoligonucleotide are modified at the 2′-position of the ribose moiety. Inone embodiment, wherein 100% of the nucleotides in said oligonucleotideare modified at the 2′-position of the ribose moiety.

The present invention further provides an oligonucleotide, wherein atleast 50% of the internucleotidic linkages are modified and at least 50%of the nucleotides in said oligonucleotide are modified at the2′-position of the ribose moiety. In one embodiment, wherein at least60% of the internucleotidic linkages are modified and at least 60% ofthe nucleotides in said oligonucleotide are modified at the 2′-positionof the ribose moiety. In one embodiment, wherein at least 70% of theinternucleotidic linkages are modified and at least 70% of thenucleotides in said oligonucleotide are modified at the 2′-position ofthe ribose moiety. In one embodiment, wherein at least 80% of theinternucleotidic linkages are modified and at least 80% of thenucleotides in said oligonucleotide are modified at the 2′-position ofthe ribose moiety. In one embodiment, wherein all of theinternucleotidic linkages are modified and all of the nucleotides insaid oligonucleotide are modified at the 2′-position of the ribosemoiety.

The present invention further provides an oligonucleotide, wherein atleast 15% of the nucleotides in said oligonucleotide comprise 2′-OMemoieties at the 2′-position of the ribose moiety. In one embodiment,wherein at least 20% of the nucleotides in said oligonucleotide comprise2′-OMe moieties at the 2′-position of the ribose moiety. In oneembodiment, wherein at least 30% of the nucleotides in saidoligonucleotide comprise 2′-OMe moieties at the 2′-position of theribose moiety. In one embodiment, wherein at least 50% of thenucleotides in said oligonucleotide comprise 2′-OMe moieties at the2′-position of the ribose moiety. In one embodiment, wherein at least60% of the nucleotides in said oligonucleotide comprise 2′-OMe moietiesat the 2′-position of the ribose moiety. In one embodiment, wherein atleast 70% of the nucleotides in said oligonucleotide comprise 2′-OMemoieties at the 2′-position of the ribose moiety. In one embodiment,wherein at least 80% of the nucleotides in said oligonucleotide comprise2′-OMe moieties at the 2′-position of the ribose moiety. In oneembodiment, wherein at least 90% of the nucleotides in saidoligonucleotide comprise 2′-OMe moieties at the 2′-position of theribose moiety. In one embodiment, wherein all of the nucleotides in saidoligonucleotide comprise 2′-OMe moieties at the 2′-position of theribose moiety.

Misc. Characteristics

The present invention further provides an oligonucleotide, wherein saidoligonucleotide is a concatemer consisting of two or moreoligonucleotide sequences joined by a linker.

The present invention further provides an oligonucleotide, wherein saidoligonucleotide is linked or conjugated at one or more nucleotideresidues, to a molecule modifying the characteristics of theoligonucleotide to obtain one or more characteristics selected from thegroup consisting of higher stability, lower serum interaction, highercellular uptake, higher viral protein interaction, an improved abilityto be formulated for delivery, a detectable signal, higher antiviralactivity, better pharmacokinetic properties, specific tissuedistribution, lower toxicity.

The present invention further provides an oligonucleotide, wherein saidoligonucleotide is double stranded.

The present invention further provides an oligonucleotide, wherein saidoligonucleotide is double or single stranded and comprises at least onebase which is capable of hybridizing via non-watson-crick interactions.

The present invention further provides an oligonucleotide, wherein saidoligonucleotide comprises a portion complementary to a viral mRNA.

The present invention further provides an oligonucleotide, wherein saidoligonucleotide binds to one or more viral components.

The present invention further provides an oligonucleotide, wherein saidoligonucleotide interacts with one or more host components, wherein saidinteraction results in inhibition of viral activity or production.

The present invention further provides an oligonucleotide, wherein atleast a portion of the sequence of said oligonucleotide is derived froma viral genome.

The present invention further provides an oligonucleotide, wherein atleast a portion of the sequence of said oligonucleotide is derived froma viral genome and has an antiviral activity that is predominantly anon-sequence complementary mode of action.

The present invention further provides an oligonucleotide, wherein atleast a portion of the sequence of said oligonucleotide is derived froma viral packaging sequence or other viral sequence involved in anaptameric interaction.

The present invention further provides an oligonucleotide, wherein atleast a portion of the sequence of said oligonucleotide is involved inan aptameric interaction with a viral component or a host component orboth.

Activity Levels

The present invention further provides an oligonucleotide, wherein saidoligonucleotide has an IC₅₀ for a target virus of 0.10 μm or less. Inone embodiment, wherein said oligonucleotide has an IC₅₀ for a targetvirus of 0.05 μm or less. In one embodiment, wherein saidoligonucleotide has an IC₅₀ for a target virus of 0.025 μm or less. Inone embodiment, wherein said oligonucleotide has an IC₅₀ for a targetvirus of 0.015 μm or less.

Target Viruses

The present invention further provides an oligonucleotide, wherein saidoligonucleotide targets a DNA virus. In one embodiment, an RNA virus. Inone embodiment, a member of the herpesviridae. In one embodiment, HSV-1.In one embodiment, HSV-2. In one embodiment, CMV. In one embodiment, amember of the hepadnaviridae In one embodiment, HBV. In one embodiment,a member of the parvoviridae. In one embodiment, a member of thepoxviridae. In one embodiment, a member of the papillomaviridae. In oneembodiment, a member of the adenoviridae In one embodiment, a member ofthe retroviridae In one embodiment, HIV-1. In one embodiment, HIV-2 Inone embodiment, a member of the paramyxoviridae. In one embodiment, RSV.In one embodiment, parainfluenza virus. In one embodiment, a member ofthe bunyaviridae. In one embodiment, hantavirus In one embodiment, amember of the picornaviridae In one embodiment, coxsackievirus. In oneembodiment, rhinovirus. In one embodiment, a member of the flaviviridaeIn one embodiment, yellow fever virus In one embodiment, dengue virus.In one embodiment, West Nile virus In one embodiment, hepatitis C virus.In one embodiment, a member of the filoviridae. In one embodiment, Ebolavirus In one embodiment, Marburg virus In one embodiment, a member ofthe orthomyxoviridae. In one embodiment, influenza virus. In oneembodiment, a member of the togaviridae. In one embodiment, a member ofthe coronaviridae. In one embodiment, a member of the reoviridae. In oneembodiment, a member of the rhabdoviridae. In one embodiment, a memberof the arenaviridae. In one embodiment, a member of the calciviridae. Inone embodiment, Varicella Zoster Virus. In one embodiment, Epstein BarrVirus. In one embodiment, Herpesvirus 6A or 6B. In one embodiment, amember of hepadnaviridae. In one embodiment, human metapneumovirus. Inone embodiment, Rift Valley fever virus. In one embodiment, CrimeanCongo Hemorrhagic Fever virus. In one embodiment, Western EquineEncephalitis virus. In one embodiment, lassa fever virus.

Oligonucleotide

The present invention further provides an oligonucleotide comprising atleast 20 linked nucleotides, wherein at least 80% of the linkages aremodified; and at least 80% of the nucleotides comprise 2′-modificationsof the ribose sugar moiety. In one embodiment, this oligonucleotide hasan antiviral activity.

In one embodiment, wherein at least 90% of the internucleotidic linkagesare modified In one embodiment, wherein all of the internucleotidiclinkages are modified. In one embodiment, wherein at least 90% of thenucleotides comprise 2′-modifications of the ribose sugar. In oneembodiment, wherein all of the nucleotides comprise 2′-modifications ofthe ribose sugar.

The present invention further provides an oligonucleotide, wherein said2′-modifications are 2′-OMe substitutions. In one embodiment, wherein atleast 90% of the nucleotides comprise 2′-OMe substitutions. In oneembodiment, wherein all of the nucleotides comprise 2′-OMesubstitutions.

The present invention further provides an oligonucleotide, wherein said2′-modifications are 2′-methoxyethoxy substitutions. In one embodiment,at least 15% of the nucleotides comprise 2′-methoxyethoxy substitutions.In one embodiment, at least 50% of the nucleotides comprise2′-methoxyethoxy substitutions. In one embodiment, at least 90% of thenucleotides comprise 2′-methoxyethoxy substitutions. In one embodiment,all of the nucleotides comprise 2′-methoxyethoxy substitutions.

The present invention further provides an oligonucleotide, wherein saidoligonucleotide is at least 40 nucleotides in length. In one embodiment,at least 50 nucleotides in length. In one embodiment, at least 60nucleotides in length. In one embodiment, at least 80 nucleotides inlength

The present invention further provides an oligonucleotide, wherein saidoligonucleotide is 30-40 nucleotides in length. In one embodiment, 40-50nucleotides in length. In one embodiment, 50-60 nucleotides in length.In one embodiment, 60-70 nucleotides in length. In one embodiment, 70-80nucleotides in length.

The present invention further provides an oligonucleotide, wherein saidoligonucleotide is free from self-complementary sequences of greaterthan 5 contiguous nucleotides. In one embodiment, greater than 10contiguous nucleotides. In one embodiment, greater than 20 contiguousnucleotides.

The present invention further provides an oligonucleotide, wherein saidoligonucleotide is free of catalytic activity.

Chain Moiety Limitations

The present invention further provides an oligonucleotide, furthercomprising 1-4 non-nucleotide chain moieties.

Mixtures

The present invention further provides an oligonucleotide mixture,comprising a mixture of at least two different antiviraloligonucleotides of the invention. In one embodiment, at least tendifferent antiviral oligonucleotides. In one embodiment, at least 100different antiviral oligonucleotides. In one embodiment, at least 1000different antiviral oligonucleotides. In one embodiment, at least 106different antiviral oligonucleotides.

The present invention further provides a mixture, wherein a plurality ofsaid different oligonucleotides are at least 10 nucleotides in length.In one embodiment, at least 20 nucleotides in length. In one embodiment,at least 30 nucleotides in length. In one embodiment, at least 40nucleotides in length. In one embodiment, at least 50 nucleotides inlength. In one embodiment, at least 60 nucleotides in length. In oneembodiment, at least 70 nucleotides in length. In one embodiment, atleast 80 nucleotides in length. In one embodiment, at least 120nucleotides in length.

Pharmaceutical Compositions

The present invention further provides an antiviral pharmaceuticalcomposition comprising a therapeutically effective amount of at leastone pharmacologically acceptable, antiviral oligonucleotide,polypyrimidine or oligonucleotide mixture, wherein the antiviralactivity of said oligonucleotide or the oligonucleotides in said mixtureoccurs principally by a sequence independent mode of action; and apharmaceutically acceptable carrier.

The present invention further provides an antiviral pharmaceuticalcomposition, adapted for the treatment, control, or prevention of adisease with a viral etiology.

The present invention further provides an antiviral pharmaceuticalcomposition, adapted for the treatment, control or prevention of a priondisease.

The present invention further provides an antiviral pharmaceuticalcomposition, adapted for delivery by a mode selected from the groupconsisting of intraocular, oral ingestion, enterally, inhalation,cutaneous injection, subcutaneous injection, intramuscular injection,intraperitoneal injection, intrathecal injection, intratrachaelinjection, and intravenous injection.

The present invention further provides an antiviral pharmaceuticalcomposition, wherein said composition further comprises a deliverysystem. In one embodiment, said delivery system targets specific cellsor specific tissues. In one embodiment, said composition furthercomprises at least one other antiviral drug in combination. In oneembodiment, said composition further comprises a non-nucleotideantiviral polymer in combination. In one embodiment, said compositionfurther comprises an antiviral antisense oligonucleotide in combination.In one embodiment, said comoposition further comprises an antiviralRNAi-inducing oligonucleotide. In one embodiment, said antiviralRNAi-inducing oligonucleotide is an siRNA.

The present invention further provides an antiviral pharmaceuticalcomposition, wherein said composition has an IC₅₀ for a target virus of0.10 μM or less. In one embodiment, an IC₅₀ for a target virus of 0.05μM or less. In one embodiment, an IC₅₀ for a target virus of 0.025 μM orless. In one embodiment, an IC₅₀ for a target virus of 0.015 μM or less.

Kits

The present invention further provides a kit comprising at least oneantiviral oligonucleotide, mixture, or antiviral pharmaceuticalcomposition in a labeled package, wherein the antiviral activity of saidoligonucleotide occurs principally by a non-sequence complementary modeof action and the label on said package indicates that said antiviraloligonucleotide can be used against at least one virus.

The present invention further provides a kit, wherein said kit containsa mixture of at least two different antiviral oligonucleotides.

The present invention further provides a kit approved by a regulatoryagency for use in humans.

The present invention further provides a kit approved by a regulatoryagency for use in at least one non-human animal. In one embodiment, saidnon-human animal is a primate In one embodiment, said non-human animalis a feline In one embodiment, said non-human animal is a bovine. In oneembodiment, said non-human animal is an ovine. In one embodiment, saidnon-human animal is a canine In one embodiment, said non-human animal isa porcine. In one embodiment, said non-human animal is an equine.

Method of Treatment

The present invention further provides a method for the prophylaxis ortreatment of a viral infection in a subject, comprising administering toa subject in need of such a treatment a therapeutically effective amountof at least one pharmacologically acceptable oligonucleotide of theinvention.

The present invention further provides use of at least oneoligonucleotide according to the invention, or pharmaceuticalcomposition according to the invention in the manufacture of amedicament for the prophylaxis or treatment of a viral infection in asubject.

In one embodiment, said subject is a human. In one embodiment, saidsubject is a non-human animal. In one embodiment, said non-human animalis a primate. In one embodiment, said non-human animal is a feline. Inone embodiment, said non-human animal is a bovine. In one embodiment,said non-human animal is an ovine. In one embodiment, said non-humananimal is a canine. In one embodiment, said non-human animal is aporcine. In one embodiment, said non-human animal is an equine. In oneembodiment, said subject is a plant.

The present invention further provides use of at least oneoligonucleotide according to the invention, or pharmaceuticalcomposition according to the invention in the manufacture of amedicament for the prophylactic treatment of cancer caused byoncoviruses in a human or a non-human animal.

In one embodiment, said oligonucleotide is administered to a human. Inone embodiment, said oligonucleotide is administered to a non-humananimal. In one embodiment, said non-human animal is a primate. In oneembodiment, said non-human animal is a feline. In one embodiment, saidnon-human animal is a bovine. In one embodiment, said non-human animalis an ovine. In one embodiment, said non-human animal is a canine. Inone embodiment, said non-human animal is a porcine. In one embodiment,said non-human animal is an equine.

Polypyrimidine Oligo-Related

The present invention further provides an oligonucleotide comprising atleast 50% of pyrimidine residues. In one embodiment, at least 80%. Inone embodiment, at least 90%. In one embodiment, only pyrimidineresidues.

The present invention further provides an oligonucleotide wherein thepyrimidine residues are cytosine residues. In one embodiment, thymineresidues. In one embodiment, cytosine or thymine residues.

The present invention further provides an antiviral pharmaceuticalcomposition comprising a therapeutically effective amount of at leastone pharmacologically acceptable, polypyrimidine oligonucleotide orpolypyrimidine oligonucleotide mixture, wherein the antiviral activityof said oligonucleotide or the oligonucleotides in said mixture occursprincipally by a sequence independent mode of action; and apharmaceutically acceptable carrier. In one embodiment, saidoligonucleotide comprises at least one modified internucleotidiclinkage.

In one embodiment, said composition is adapted for administration to anacidic in vivo site.

In one embodiment, said composition further comprises a penetrationenhancer.

In one embodiment, said composition further comprises a surfactant.

In one embodiment, said composition is in the form of a powder.

In one embodiment, said composition is in the form of granules.

In one embodiment, said composition is in the form of microparticulates.

In one embodiment, said composition is in the form of nanoparticulates.

In one embodiment, said composition is in the form of a suspension orsolution.

In one embodiment, said composition is in the form of an emulsion.

In one embodiment, said composition is adapted for oral administration.

In one embodiment, wherein said composition is adapted for vaginaladministration.

In one embodiment, said composition comprises at least one polyColigonucleotide.

In one embodiment, said composition comprises at least one polyToligonucleotide.

In one embodiment, said composition comprises at least one polyCToligonucleotide.

In one embodiment, said composition is approved for administration to ahuman.

In one embodiment, said composition is approved for administration to amammal.

In one embodiment, said composition is approved for administration to anon-mammal animal.

The present invention further provides use of a pharmaceuticalcomposition adapted for administration to an acidic in vivo site,wherein said composition contains at least one pharmacologicallyacceptable polypyrimidine oligonucleotide in the manufacture of amedicament for the prophylaxis or treatment of a viral infection in asubject.

In one embodiment, said subject is a human. In one embodiment, saidsubject is a mammal. In one embodiment, said subject is a non-mammalanimal.

As described in the Background, a number of antisense oligonucleotides(ONs) have been tested for antiviral activity. However, such antisenseONs are sequence-specific, and typically are about 16-20 nucleotides inlength.

As demonstrated by the results in Examples 1 and 2, the antiviral effectof random PS-ONs is not sequence specific. Considering the volumes andconcentrations of PS-ONs used in those tests, it is almost theoreticallyimpossible that a particular random sequence is present at more than 1copy in the mixture. This means than there can be no antisense effect inthese PS-ON randomers. In the latter example, should the antiviraleffect be caused by the sequence-specificity of the PS-ONs, such effectwould thus have to be caused by only one molecule, a result that doesnot appear possible. For example, for an ON randomer 40 bases in length,any particular sequence in the population would theoretically representonly ¼⁴⁰ or 4.1×10⁻⁴¹ of the total fraction. Given that 1mole=6.022×10²³ molecules, and the fact that our largest synthesis iscurrently done at the 15 micromole scale, all possible sequences willnot be present and also, each sequence is present most probably as onlyone copy. Of course, one skilled in the art applying the teaching of thepresent invention could also use ONs that have sequences of suchsequence specific ONs, but utilize the sequence independent activitydiscovered in the present invention. Accordingly, the present inventionis not to be restricted to non-sequence complementary ONs, but disclaimswhat has been disclosed in the prior art regarding sequence-specificantisense and RNAi (e.g., siRNA) ONs for treating viral infections.

For applicable viruses (including, for example, those for which data isdescribed herein), as the size of the randomer increases, so does itsantiviral potency for lengths up to and even exceeding 40 nucleotides.It should be pointed out that due to limitations in currentphosphoramidite-based oligonucleotide synthesis, the larger PS-ONs(e.g., 80- and 120-mers) have a significant contamination of fragmentssmaller than the desired size. The weaker effects (on a per base basis)seen with larger oligos (80 and 120 bp) may reflect the lowerconcentration of full-length randomers in these populations and may alsoreflect a decreased availability at the appropriate site. It may bepossible to achieve much larger increases in antiviral activity iflarger randomers (>40 bases) of reasonable purity (e.g., at least 75%full length) are synthesized or purified, and/or if the delivery of anyof these ONs is facilitated by a delivery system, e.g., a deliverysystem providing targeting or sustained release.

In the present invention, randomers (or other antiviral oligonucleotidesas described herein) may block viral replication by several mechanisms,including but not limited to the following: 1. preventing the adsorptionor receptor interaction of virions, thus preventing infection, 2. dopingthe virus assembly or the packaging of viral genomes into capsids(competing with viral DNA or RNA for packaging), resulting in defectivevirions, 3. disrupting and or preventing the formation of capsids duringpackaging or the interaction of capsid proteins with other structuralproteins, resulting in inhibition of viral release or causing therelease of defective virions, 4. binding to key viral components andpreventing or reducing their activity, 5. binding to key host componentsrequired for viral proliferation.

Without being limited on the mechanism by which the present viralinhibition is achieved, as indicated above there are several possiblemechanisms that could explain and/or predict the inhibitory propertiesof ONs against viral replication. The first of these is that the generalaptameric effect of ONs is allowing for their attachment, either toproteins on the cell surface or to viral proteins, preventing viraladsorption and fusion. The size threshold for effect may be a result ofa certain cumulative charge required for interaction.

A second possible mechanism is that ONs may function within the cell bypreventing packaging and/or assembly of the virus. ONs above a certainsize threshold may compete or interfere with the normal capsid/nucleicacid interaction, preventing the packaging of a functional viral genomeinside new viruses. Alternatively, ONs may prevent the formation of anormal capsid, which could prevent normal viral budding, alter viralstability, or prevent proper virion disassembly upon internalization.

While the mechanism of action is not yet entirely clear, assay resultsdemonstrate that the present ONs can exhibit greater efficacy in viralinhibition compared to the clinical correlates, acyclovir, gancyclovir,Ribavirin, and protease inhibitors. ONs in accordance with the presentinvention could thus be used for treating or preventing viral infection.The viral infections treated could be those caused by human, animal, andplant viruses

Chemical modification of oligonucleotides can advantageously be used toenhance the stability and/or activity of the present antiviraloligonucleotides. Methoxylation and other modifications at the2′-position of the ribose on RNA have been shown to render RNA stable tonucleases, to minimize the protein binding observed withphosphorothioated nucleic acids and to increase the melting temperatureof these oligos with their target sequences. While 2′-O methylation andother 2′-modifications are currently used to improve the characteristicsof antisense oligonucleotides, oligonucleotides with such modficationsdo not elicit RNase H activity when present on every ribose, makingcompletely 2′-modified oligonucleotides poorer candidates for antisenseactivity. This has resulted in the use of 2′-O methyl and other 2′modification “gapmers” which contain 2′ modifications only at theextremities of the oligonucleotide, thus retaining the ability of theoligo to activate RNase H. To our knowledge, there is no report of anon-sequence specific antiviral oligonucleotide with phosphorothioatelinkages and ribonucleotides such as 2′-O-methyl or other 2′modification on each ribose sugar in the oligonucleotide.

As described herein, we had found that the 40 base PS-ON randomer is apotent inhibitor of several different viruses. We suggest thenon-limiting hypothesis that the thioated backbone imparts an increasedhydrophobic character to the ON randomer, which may allow it to interactwith hydrophobic domains in viral fusion proteins. These hydrophobicdomains are believed to be essential for the membrane fusion activity ofmany different viruses including HSV, HIV, influenza, RSV, and Ebola. Inthe case of HIV, such hydrophobic domain has been used as a target forthe development of fusion inhibitors.

Thus, the incorporation of phosphorothioate linkages and ribonucleotidemodifications, including 2′-O-methyl and other 2′ sugar modifications,into oligonucleotides of this invention, is useful for improvingcharacteristics of non-sequence specific antiviral oligonucleotides.Results demonstrate that modification at the ′2-position of each riboseof PS-ONs does not significantly alter their antiviral activity, butthat such modification reduces the general interaction of the PS-ONswith serum proteins and renders them significantly more resistant to lowpH. These properties promise to increase the pharmacokinetic performanceand reduce the toxic side effects normally seen with PS-ONs. Forexample, their pH resistance make them more suitable for oral delivery.Also their lowered interaction with serum proteins promises to improvetheir pharmacokinetic behaviour without affecting their antiviralactivity. Thus, oligonucleotides having each linkage phosphorothioatedand each ribonucleotide modified at the 2′-position of the ribose, e.g.,2′-O-methyl modifications, have antiviral activity but do not triggerRNase H activity, a property desirable for traditional antisenseoligonucleotide but completely dispensable for the activity described inthis present invention. Results also demonstrate that modifications atthe ′2-position of each ribose of PS-ONs renders the ON more resistantto nucleases in comparison with a PS-ON comprising the samemodifications but only at both ends (gapmer). Gapmers are preferentiallyused in the antisense technology. Nuclease resistance of PS-ONsincluding modifications at the ′2-position of each ribose should displaybeneficial properties, such as improved pharmakokinetics and/or oralavailability.

In addition, while PS-ONs that include modifications at the 2′-positionof each ribose show desirable characteristics, PS-ONs with substantialnumbers of modifications at the 2′-position of riboses would alsodisplay desirable characteristic, e.g., modification at at least 50% ofthe riboses and more preferably 80% or even more.

As described above, the activity of the present oligonucleotides doesnot target any nucleic acid by hybridization since randomers, forexample, have no antisense activity. Thus, we believe that theoligonucleotides target proteins. Since the addition of 2′-O-methylribose modifications to phosphorothioate oligonucleotides lowers theprotein binding activity (Kandimalla et al., 1998, Bioorganic Med ChemLett. 8:2103-2108; Mou et al., 2002, Nucleic Acids Res. 30:749-758), itwould be expected that these modifications would lower antiviralactivity. Unexpectedly, we found that addition of 2′-O-methyl ribosemodifications to phosphorothioate oligonucleotides does not affect theantiviral activity.

Assay results for a number of different oligonucleotides are describedherein. Unless otherwise indicated, the tested oligonucleotides have2′-H moieties (2′-deoxy) and are thus ODNs. However, the sequenceindependent activities of the present invention are not limited tooligonucleotides with such 2′-H moieties, but is also present for oligoscontaining nucleotides having 2′-OH moieties as well as other2′-modifications, for example, 2′-O-methyl and 2′-fluoro.

The description herein utilizes a number of abbreviations, including thefollowing:

Selected Abbreviations

ON: Oligonucleotide

ODN: Oligodeoxynucleotide

PS: Phosphorothioate

PS2: Phosphorodithioate

PRA: Plaque reduction assay

PFU: Plaque forming unit

INF A: Influenza A virus

HIV: Human immunodeficiency virus (includes both HIV-1 and HIV-2 if notspecified)

HSV: Herpes simplex virus (includes both HSV-2 and HSV-3 if notspecified)

RSV: Respiratory syncytial virus

COX: Coxsackievirus

DHBV: Duck hepatitis B virus

Broad Spectrum Antiviral Activity

According to the conclusions discussed above and the data reportedherein, it appeared that random ONs and ON randomers could havebroad-spectrum antiviral activity with viruses where assembly and/orpackaging and/or encapsidation of the viral genome is a required step inreplication. Therefore to test this hypothesis, several PS-ON randomersof different sizes were selected to be tested in cellular models ofvarious viral Infections. A number of such tests are described herein inthe Examples, including tests with CMV, HIV-1, RSV, Coxsackie virus B2,DHBV, Hantavirus, Parainfluenza virus, and Vaccinia virus, as well asthe tests on HSV-1 and HSV-2 described in Examples 1 and 2.

Conclusions on Broad Spectrum Antiviral Activity

The efficacy studies with different viruses demonstrate that random ONsand randomers display inhibitory properties against a variety ofdifferent viruses. Moreover, these studies support the conclusion thatlarger randomers display greater efficacy for viral inhibition thansmaller randomers. This suggests a common size and/or charge dependentmechanism for the random ONs or ON randomers activity in allencapsidating viruses.

While HSV and CMV are both double-stranded DNA viruses of theherpesviridae family, HIV is a RNA virus from the retroviridae, and RSVa RNA virus from the paramyxoviridae. Given the fact that ON randomerscan inhibit viral function in a variety of different viruses, withoutbeing limited to the mechanisms listed, as discussed above the followingmechanisms are reasonable: A) ONs/ON randomers are inhibiting viralinfection via an aptameric effect, preventing viral fusion with theplasma membrane; and/or B) ONs/ON randomers are preventing or doping theassembly of virions or the packaging of viral DNA within capsidsresulting in defective virions; and/or C) ONs/ON randomers areinterfering with host proteins or components required in the assemblyand/or packaging and/or gene expression of the virus.

Requirement for Antiviral Activity

Since a randomized DNA sequence seems to be sufficient for viralinhibition, it was interesting to see if antiviral activity could bemaintained in the absence of the phosphorothioate modification and alsoif the efficacy was augmented by either choosing a random sequence or aspecific sequence found in the viral genome.

Accordingly, DNA and RNA modifications were investigated with respect totheir effect on the antiviral efficacy of the randomers. Since randomerswork via a sequence independent, e.g., non-sequence complementary,mechanism, these experiments were designed to test the slight changes innucleic acid conformation and charge distribution on antiviral efficacy.

To test if ONs with different nucleotide/nucleoside modifications couldinhibit HSV-1, REP 2024, 2026, 2059, and 2060 were tested in the HSV-1PRA as described in the Examples. REP 2024 (a PS-ON with a 2-0-Methylmodification to the ribose on 4 bases at both termini of the ON), REP2026 (a PO-ON with methylphosphonate modifications to the linkagesbetween the 4 bases at both termini of the ON), REP 2059 (RNA PS-ONrandomer 20 bases in length), and REP 2060 (RNA PS-ON randomer 30 basesin length) showed anti-HSV-1 activity. The assay was conducted as aplaque reduction assay in VERO cells using HSV-1 (strain KOS). ThePS-ONs were tested in increasing concentrations. IC₅₀ values calculatedfrom linear regressions were 0.14, 3.41, 1.36, and 0.80 respectively.

In the latter example, should the antiviral effect be caused only by theONs consisting of DNA phosphorothioate backbone, such effect would thusbe caused by only one molecule. But other backbones and modificationsgave positive antiviral activity. Of course, one skilled in the artapplying the teaching of the present invention could also use differentchemistry ONs. A modification of the ON, such as, but not limited to, aphosphorothioate modification, appears to be beneficial for antiviralactivity. This is most likely due to the needed charge of ONs and/or therequirement for stabilization of DNA both in the media andintracellularly, and it may also be due to the chirality of the PS-ONs.

Compound REP 2026 showed an antiviral activity while having a centralportion comprising unmodified PO-nucleotides and 4 methylphosphonatelinkages at both termini protecting from degradation. This indicatesthat PO-ONs can be used as antivirals while protected from degradation.This protection can be achieved by modifying nucleotides at terminiand/or by using a suitable delivery system as described later.

In general, the sequence composition of the DNA used has little effecton the overall efficacy, whether randomer, random sequence or a specificHSV-1 sequence. However, at intermediate lengths, HSV-1 sequence wasalmost 3× more potent than a random sequence. This data suggests thatwhile specific antisense functionality exists for specific HSVsequences, sequence independent mechanism (the non-antisense mechanism)elucidated herein may represent the predominant part of this activity.Indeed, as the ON grows to 40 bases, essentially all of the antiviralactivity can be attributed to a sequence independent (e.g.,non-antisense) effect.

Lower Toxicity of Randomer

One goal of using an ON randomer is to lower the toxicity. It is knownthat different sequences may trigger different responses in the animal,such as general toxicity, interaction with serum proteins, andinteraction with immune system (Monteith et al (1998) Toxicol Sci46:365-375). The mixture of ONs may thus decrease toxic effects becausethe level of any particular sequence will be very low, so that nosignificant interaction due to sequence or nucleotide composition islikely.

Pharmaceutical Compositions

The ONs of the invention may be in the form of a therapeutic compositionor formulation useful for treating (or prophylaxis of) viral diseases,which can be approved by a regulatory agency for use in humans or innon-human animals, and/or against a particular virus or group ofviruses. These ONs may be used as part of a pharmaceutical compositionwhen combined with a physiologically and/or pharmaceutically acceptablecarrier. The characteristics of the carrier may depend on the route ofadministration. The pharmaceutical composition of the invention may alsocontain other active factors and/or agents which enhance activity.

Administration of the ONs of the invention used in the pharmaceuticalcomposition or formulation or to practice the method of treating ananimal can be carried out in a variety of conventional ways, such asintraocular, oral ingestion, enterally, inhalation, or cutaneous,subcutaneous, intramuscular, intraperitoneal, intrathecal,intratracheal, or intravenous injection.

The pharmaceutical composition or oligonucleotide formulation of theinvention may further contain other chemotherapeutic drugs for thetreatment of viral diseases, such as, without limitation, Rifampin,Ribavirin, Pleconaryl, Cidofovir, Acyclovir, Pencyclovir, Gancyclovir,Valacyclovir, Famciclovir, Foscarnet, Vidarabine, Amantadine, Zanamivir,Oseltamivir, Resquimod, antiproteases, pegylated interferon (Pegasys™)anti HIV proteases (e.g. lopinivir, saquinivir, amprenavir, HIV fusioninhibitors, nucleotide HIV RT inhibitors (e.g., AZT, Lamivudine,Abacavir), non-nucleotide HIV RT inhibitors, Doconosol, Interferons,Butylated Hydroxytoluene (BHT) and Hypericin. Such additional factorsand/or agents may be included in the pharmaceutical composition, forexample, to produce a synergistic effect with the ONs of the invention.

The pharmaceutical composition or oligonucleotide formulation of theinvention may further contain a polymer, such as, without restriction,polyanionic agents, sulfated polysaccharides, heparin, dextran sulfate,pentosan polysulfate, polyvinylalcool sulfate, acemannan,polyhydroxycarboxylates, cellulose sulfate, polymers containingsulfonated benzene or naphthalene rings and naphthalene sulfonatepolymers, acetyl phthaloyl cellulose, poly-L-lysine, sodium caprate,cationic amphiphiles, cholic acid. Polymers are known to affect theentry of virions in cells by, in some cases, binding or adsorbing to thevirion itself. This characteristic of antiviral polymers can be usefulin competing with ONs for the binding, or adsorption to the virion, theresult being an increased intracellular activity of the ONs compared toits extracellular activity.

Exemplary Lipid Encapsulation and Delivery

Although PS-ONs (as well as oligonucleotides with other modifiedlinkages) are more resistant to endogenous nucleases than naturalphosphodiesters, they are not completely stable and are slowly degradedin blood and tissues. A limitation in the clinical application of PSoligonucleotide drugs is their propensity to activate complement on i.v.administration. In general, liposomes and other delivery systems enhancethe therapeutic index of drugs, including ONs, by reducing drugtoxicity, increasing residency time in the plasma, and delivering moreactive drug to disease tissue by extravasation of the carriers throughhyperpermeable vasculature. Moreover in the case of PS-ON, lipidencapsulation prevents the interaction with potential protein-bindingsites while in circulation (Klimuk et al. (2000) J Pharmacol Exp Ther292:480-488).

According to our results described herein, an approach is to use adelivery system such as, but without restriction, lipophilic molecules,polar lipids, liposomes, monolayers, bilayers, vesicles, programmablefusogenic vesicles, micelles, cyclodextrins, PEG, iontophoresis, powderinjection, and nanoparticles (such as PIBCA, PIHCA, PHCA, gelatine,PEG-PLA) for the delivery of ONs described herein and/or antisense andsiRNA oligonucleotides. Use of such delivery systems can, withoutlimitation, provide one or more of the following benefits: lower thetoxicity of the active compound in animals and humans, lower the IC₅₀,increase the duration of action from the standpoint of drug delivery,and protect the oligonucleotides from non-specific binding with serumproteins.

Thus, we have shown that the antiviral activity of PS-ON randomersincreases with increasing size. Moreover this activity is correlatedwith increased affinity for viral proteins (in a viral lysate). Since itis well known in the art that the phosphorothioate modificationincreases the affinity of protein-DNA interaction, we tested the abilityof increasingly larger PS-ON randomers to bind to fetal bovine serum(FBS) using the same FP-based assay used for measuring interaction withviral lysates. In this assay, 250 ug of non-heat inactivated FBS wascomplexed with a fluorescently labeled 20 base PS-ON randomer, underconditions where the binding (mP value) was saturated. Unlabelled PS-ONrandomers of increasing size (REP 2003, REP 2004, REP 2006 and REP 2007)were used to compete the interaction of FBS with the labeled bait. Theresults of this test clearly show that as the size of the PS-ON randomerincreases, so does its affinity for FBS. This result suggests that themost highly active anti-viral PS-ONs will also be the ones to bind withthe highest affinity to proteins.

However, it is known in the art that one of the main therapeuticproblems for phosphorothioate antisense oligonucleotides is their sideeffects due mainly to an increased interaction with proteins(specifically with serum proteins) as described by Kandimalla andco-workers (Kandimalla et al. (1998) Bioorg. Med. Chem. Lett.8:2103-2108). Therefore, in some cases it may be beneficial to use asuitable delivery system capable of delivering antiviral ONs to the siteof action while preventing their interaction with serum proteins. Inaddition, it may be beneficial to use suitable delivery systems forcombination use of the present sequence independent ONs with other typesof ant-viral ONs such as antisense oligonucleotides and siRNAs.

To demonstrate certain effects of a delivery system, we tested twodifferent delivery technologies which are liposomal based; Cytofectinand DOTAP. We measured the protection of REP2006 from serum proteininteractions by DOTAP and Cytofectin in our in vitro FP-basedinteraction assay. Unencapsulated REP 2006 was able to compete boundfluorescent oligo from serum but when REP 2006 was encapsulated witheither DOTAP or Cytofectin it was no longer able to compete for serumbinding. These data suggest that encapsulation protects oligos fromserum interaction and will result in better pharmacokinetic behaviourwith fewer side effects.

We also measured the delivery of the PS-ON randomer REP 2006(encapsulated with either Cytofectin or DOTAP) into 293A cells in thepresence of high concentrations of serum (50%) by measuring theintracellular concentration of labeled REP 2006 by fluorometry. Theseresults show that such delivery agents increase the intracellularconcentration of REP 2006, and also that, in the case of DOTAP, thelevels of intracellular REP 2006 after 24 hours were markedly increased.Finally, we measured the protection of REP2006 from serum proteininteractions by DOTAP and cytofectin in our in vitro FP-basedinteraction assay. Unencapsulated REP 2006 was able to compete boundfluorescent oligo from serum but when REP 2006 was encapsulated witheither DOTAP or cytofectin it was no longer able to compete for serumbinding. These data suggest that encapsulation protects oligos fromserum interaction and will result in better pharmacokinetic behaviourwith fewer side effects.

Similarly demonstrating the effect of lipid encapsulation ofoligonucleotides, we monitored the uptake of an additional PS-ONrandomer by exposing cultured cells to fluorescently labeled randomersand then examined the fluorescence intensity in lysed cells after tworounds of washing. The cellular uptake of cells exposed to 250 nM REP2004-FL was tested with no delivery and after encapsulation in one ofthe following lipid based delivery systems; Lipofectamine™ (Invitrogen),Polyfect™ (Qiagen) and Oligofectamine™ (Invitrogen). After 4 hours,cells were washed twice with PBS and lysed using MPER lysis reagent(PROMEGA). The relative fluorescence yield from equivalent numbers ofexposed cells with and without lipid system was detected. We observedthat in the presence of all three agents tested, there was a significantincrease in the intracellular PS-ON concentration compared to nodelivery.

In keeping with the test results, the use of a delivery system can serveto protect oligonucleotides from serum interactions, reducing sideeffects and increasing tissue distribution and/or can significantlyincrease the intracellular delivery of ONs.

Another potential benefit in using a delivery system is to protect theONs from interactions, such as adsorption, with infective virions inorder to prevent amplification of viral infection through differentmechanisms such as increased cellular penetration of virions.

Another approach is to accomplish cell specific delivery by associatingthe delivery system with a molecule(s) that will increase affinity withspecific cells, such molecules being without restriction antibodies,receptor ligands, vitamins, hormones and peptides.

Additional options for delivery systems are provided below.

Linked ON

In certain embodiments, ONs of the invention are modified in a number ofways without compromising their ability to inhibit viral replication.For example, the ONs are linked or conjugated, at one or more of theirnucleotide residues, to another moiety. Thus, modification of theoligonucleotides of the invention can involve chemically linking to theoligonucleotide one or more moieties or conjugates which enhance theactivity, cellular distribution, increase transfer across cellularmembranes specifically or not, or protecting against degradation orexcretion, or providing other advantageous characteristics. Suchadvantageous characteristics can, for example, include lower seruminteraction, higher viral-protein interaction, the ability to beformulated for delivery, a detectable signal, improved pharmacokineticproperties, and lower toxicity. Such conjugate groups can be covalentlybound to functional groups such as primary or secondary hydroxyl groups.For example, conjugate moieties can include a steroid molecule, anon-aromatic lipophilic molecule, a peptide, cholesterol,bis-cholesterol, an antibody, PEG, a protein, a water soluble vitamin, alipid soluble vitamin, another ON, or any other molecule improving theactivity and/or bioavailability of ONs.

In greater detail, exemplary conjugate groups of the invention caninclude intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, SATE, t-butyl-SATE, groups thatenhance the pharmacodynamic properties of oligomers, and groups thatenhance the pharmacokinetic properties of oligomers. Typical conjugategroups include cholesterols, lipids, phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, fluorescent nucleobases, and dyes.

Groups that enhance the pharmacodynamic properties, in the context ofthis invention, include groups that enhance oligomer resistance todegradation and/or protect against serum interaction. Groups thatenhance the pharmacokinetic properties, in the context of thisinvention, include groups that improve oligomer uptake, distribution,metabolism or excretion. Exemplary conjugate groups are described inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992,which is incorporated herein by reference in its entirety.

Conjugate moieties can include but are not limited to lipid moietiessuch as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med.Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et at.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et at., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et at.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et at., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et at, Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et at., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylaminocarbonyl-oxycholesterol moiety (Crooke et al., J. PharmacolExp. Ther., 1996, 277, 923-937.

The present oligonucleotides may also be conjugated to active drugsubstances, for example without limitation, aspirin, warfarin,phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic.

Exemplary U.S. patents that describe the preparation of exemplaryoligonucleotide conjugates include, for example, U.S. Pat. Nos.4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis incorporated by reference herein in its entirety.

Another approach is to prepare antiviral ONs as lipophilicpro-oligonucleotides by modification with enzymatically cleavable chargeneutralizing adducts such as s-acetylthio-ethyl or s-pivasloylthio-ethyl(Vives et al., 1999, Nucl Acids Res 27: 4071-4076). Such modificationshave been shown to increase the uptake of ONs into cells, and thereforeare beneficial for ONs that are active intracellularly.

Design of Non-Specific ONs

In another approach, an antiviral ON demonstrating low, preferably thelowest possible, homology with the human (or other subject organism)genome is designed. The goal is to obtain an ON that will show thelowest toxicity due to interactions with human or animal genomesequence(s) and mRNAs. The first step is to produce the desired lengthsequence of the ON, e.g., by aligning nucleotides A, C, G, T in a randomfashion, manually or, more commonly, using a computer program. Thesecond step is to compare the ON sequence with a library of humansequences such as GenBank and/or the Ensemble Human Genome Database. Thesequence generation and comparison can be performed repetitively, ifdesired, to identify a sequence or sequences having a desired lowhomology level with the subject genome. Desirably, the ON sequence is atthe lowest homology possible with the entire genome, while alsopreferably minimizing self interaction.

Non-Specific ONs with Antisense Activity

In another approach, an antiviral non-specific sequence portion(s)is/are coupled with an antisense sequence portion(s) to increase theactivity of the final ON. The non-specific portion of the ONs isdescribed in the present invention. The antisense portion iscomplementary to a viral mRNA.

Non-Specific ONs with a G-Rich Motif Activity

In another approach, an antiviral non-specific sequence portion(s)is/are coupled with a motif portion(s) to improve the activity of thefinal ON. The non-specific portion of the ON is described in the presentinvention. The motif portion can, as non-limiting examples, include,CpG, Gquartet, and/or CG that are described in the literature asstimulators of the immune system. Agrawal et al. (2001) Curr. CancerDrug Targets 3:197-209.

Non-Watson-Crick ONs

Another approach is to use an ON composed of one type or more ofnon-Watson-Crick nucleotides/nucleosides. Such ONs can mimic PS-ONs withsome of the following characteristics similar to PS-ONs: a) the totalcharge; b) the space between the units; c) the length of the chain; d) anet dipole with accumulation of negative charge on one side; e) theability to bind to proteins; f) the ability to bind viral proteins, g)the ability to penetrate cells, h) an acceptable therapeutic index, i)an antiviral activity. The ON has a preferred phosphorothioate backbonebut is not limited to it. Examples of non-Watson-Cricknucleotides/nucleosides are described in Kool, 2002, Acc. Chem. Res.35:936-943; and Takeshita et al., (1987) J. Biol. Chem. 262:10171-10179where ONs containing synthetic abasic sites are described.

Antiviral Polymer

Another approach is to use a polymer mimicking the activity ofphosphorothioate ONs. As described in the literature, several anionicpolymers were shown to have antiviral inhibitory activity. Thesepolymers belong to several classes: (1) sulfate esters ofpolysaccharides (dextrin and dextran sulfates; cellulose sulfate); (2)polymers containing sulfonated benzene or naphthalene rings andnaphthalene sulfonate polymers; (3) polycarboxylates (acrylic acidpolymers); and acetyl phthaloyl cellulose (Neurath et al. (2002) BMCInfect Dis 2:27); and (4) abasic oligonucleotides (Takeshita et al.,1987, J. Biol. Chem. 262:10171-10179). Other examples of non-nucleotideantiviral polymers are described in the literature. The polymersdescribed herein mimic PS-ONs described in this invention and have thefollowing characteristics similar to PS-ONs: a) the length of the chain;b) a net dipole with accumulation of negative charge on one side; c) theability to bind to proteins; d) the ability to bind viral protein, e) anacceptable therapeutic index, f) an antiviral activity. In order tomimic the effect of a PS-ON, the antiviral polymer may preferably be apolyanion displaying similar space between its units as compared to aPS-ON. It may also have the ability to penetrate cells alone or incombination with a delivery system.

Antiviral Activity of Double-Stranded PS-ONs

A random sequence (REP 2017) and its complement (either PS modified orunmodified) are fluorescently labeled as described elsewhere and testedfor their ability to bind to purified HSV-1 and HIV-1 proteins byfluorescence polarization as described in the present invention.Hybridization was verified by acrylamide gel electrophoresis. UnmodifiedREP 2017 (2017U), either single (ss) or double stranded (ds), had nobinding activity in either HSV-1 or HIV-1 lysates. However, PS modifiedREP 2017, either single stranded or double stranded, was capable ofHSV-1 and HIV-1 interaction.

According to our results described herein, an approach is to use doublestranded ONs as effective antiviral agents. Preferentially such ONs havea phosphorothioate backbone but may also have other and/or additionalmodifications which increase antiviral activity and/or stability and/ordelivery characteristics as described herein for single stranded ONs.

In Vitro Assay for Drug Discovery

An in vitro assay is developed based on fluorescence polarization tomeasure the ability of PS-ONs to bind to viral components, e.g., viralproteins. When a protein (or another interactor) binds to thefluorescently labeled bait, the three dimensional tumbling of the baitin solution is retarded. The retardation of this tumbling is measured byan inherent increase in the polarization of excited light from thelabeled bait. Therefore, increased polarization (reported as adimensionless measure [mP]) is correlated with increased binding.

One methodology is to use as bait a PS-ON randomer labeled at the 3′ endwith FITC using an inflexible linker (3′-(6-Fluorescein) CPG). ThisPS-ON randomer is diluted to 2 nM in assay buffer (10 mM Tris, pH7.2, 80mM NaCl, 10 mM EDTA, 100 mM b-mercaptoethanol and 1% tween 20). Thisoligo is then mixed with an appropriate interactor. In this case, we uselysates of sucrose gradient purified HSV-1 (strain MacIntyre), HIV-1(strain Mn) or RSV (strain A2) suspended in 0.5M KCl and 0.5% TritonX-100 (HSV-1 and HIV-1) or 10 mM Tris, pH7.5, 150 mM NaCl, 1 mM EDTA and0.1% Triton X-100 (RSV). Following bait interaction, the complexes arechallenged with various unlabelled PS-ONs to assess their ability todisplace the bait from its complex.

In a preliminary test with three baits of different sizes; 6 (REP2032-FL), 10 (REP 2003-FL) and 20 bases (REP 2004-FL), the baits weretested for their ability to interact with HSV-1, HIV-1, and RSV lysates.Viral lysate binding to baits of different sizes was determined byfluorescence polarization. In the presence of any of the viral lysatesthe degree of binding was dependent on the size of the bait used, with2004-FL displaying the largest shift in mP (binding) in the presence ofviral lysate. We note that this is similar to the size dependentantiviral efficacy of PS-ON randomers. This bait was then used to assessthe ability of PS-ONs of different sizes to compete the interaction ofthe bait with the lysate.

The interaction of REP 2004-FL with HSV-1, HIV-1, and RSV lysates waschallenged with PS-ONs of increasing size. Determination of affinity ofPS-ON randomers for the viral lysates was detected by fluorescencepolarization. Using REP 2004-FL as the bait, complex formation withHSV-1 lysate, HIV-1 lysate, or RSV lysate was challenged with increasingconcentrations of REP 2003, REP 2004, REP 2006 or REP 2007. For eachviral lysate tested, we note that REP 2003 is unable to compete the baitaway from the lysate. The bait interaction was very strong as revealedby the relatively weak competition elicited by the REP 2004 (unlabeledbait) competitor. However, it was observed that as the size of thecompetitor PS-ON increased above 20 bases, its ability to displace thebait became more robust. This indicates an increased affinity to proteincomponents in the viral lysate as the PS-ON randomer size increases.This phenomenon mirrors the increased antiviral activity of larger PS-ONrandomers against HSV-1, HSV-2, CMV, HIV-1 and RSV.

The similarity between the efficacy in bait competition and antiviralactivity of PS-ON randomers indicates that this assay paradigm is a goodpredictor of antiviral activity. This assay is robust, easy to performand very stable, making it a very good candidate for a high throughputscreen to identify novel antiviral molecules based not on specifictarget identification but on their ability to interact with one or morecomponents, e.g., viral proteins.

While the exemplary method described herein utilizes fluorescencepolarization to measure interaction with the viral lysate, numeroustechniques are known in the art for monitoring protein interactions, andcan be used in the present methods. These include without restrictionsurface plasmon resonance, fluorescence resonance energy transfer(FRET), enzyme linked immunosorbent assay (ELSIA), gel electrophoresis(to measure mobility shift), isothermal titration and differentialscanning microcalorimetry and column chromatography. These otherdifferent techniques can be applied to measure the interaction of ONswith a viral lysate or component, and thus can be useful in screeningfor compounds which have anti-viral activity.

The method described herein is used to screen for novel compounds fromany desired source, for example, from a library synthesized bycombinatorial chemistry or isolated by purification of naturalsubstances. It can be used to a) determine appropriate size,modifications, and backbones of novel ONs; b) test novel moleculesincluding novel polymers; predict a particular virus' susceptibility tonovel ONs or novel compounds; or d) determine the appropriate suite ofcompounds to maximally inhibit a particular virus.

The increased lysate affinity with larger sized PS-ON randomers suggeststhat the antiviral mechanism of action of PS-ON randomers is based on aninteraction with one or more viral protein components which preventseither the infection or correct replication of virions. It also suggeststhat this interaction is charge (size) dependent and not dependent onsequence. As these PS-ON randomers have a size dependent activity acrossmultiple viruses spanning several different families, we suggest thatPS-ON randomers interfere with common, charge dependent protein-proteininteractions, protein-DNA/RNA interactions, and/or othermolecule-molecule interactions. These interactions can include (but arenot limited to):

-   -   The interaction between individual capsid subunits during capsid        formation.    -   The interaction between the capsid/nucleocapsid protein and the        viral genome.    -   The interaction between the capsid and glycoproteins during        budding.    -   The interaction between glycoproteins and their receptors during        infection.    -   The interaction between other key viral components involved in        viral replication.

These multiple, simultaneous inhibitions of protein-protein interactionsrepresent a novel mechanism for antiviral inhibition.

Effect of PS-ON Sequence Composition on Viral Lysate Interaction

We monitored the ability of PS-ONs of different sequences to interactwith several viral lysates. In each case, a 20-mer PS-ON is labeled atthe 3′ end with FITC as previously described herein. The PS-ONs testedconsisted of A20, T20, G20, C20, AC10, AG10, TC10, TG10, REP 2004 andREP 2017. Each of these sequences is diluted to 4nM in assay buffer andincubated in the presence of lug of HSV-1, HIV-1 or RSV lysate.Interaction is measured by fluorescence polarization.

The profile of interaction with all sequences tested is similar in allviral lysates, indicating that the nature of the binding interaction isvery similar. The ability of 20-mer PS-ONs of different sequencecompositions (A20, C20, G20, T20, AC10, AG10, TC10, TG10, REP2004,REP2017) to bind to viral lysates was measured by fluorescencepolarization. PS-ONs 3′ labeled with FITC were incubated in the presenceof lug of HSV-1, HIV-1 or RSV lysates. Within each lysate, the PS-ONs ofuniform composition (A20, G20, T20, C20) were the weakest interactorswith A20 being the weakest interactor of these by a significant margin.For the rest of the PS-ONs tested, all of them displayed a similar,strong interaction with the exception of TG10, which consistentlydisplayed the strongest interaction in each lysate. The binding profilesfor these PS-ONs is similar in all three viral lysates.

Target Identification for PS-ON Randomers in HIV-I

The ability of PS-ON randomers to bind to purified HIV-1 proteins wastested by fluorescence polarization as described in example 9.Increasing quantities of purified HIV-1 p24 or purified HIV-1 gp41 werereacted with REP 2004-FL. We note that for both these proteins, there isa protein concentration dependent shift in fluorescence polarization,indicating an interaction with both these proteins.

The ability of a range of sizes of PS-ON randomers to bind to theseproteins was also tested using fluorescent versions of REP 2032, REP2003, REP 2004, REP 2006 and REP 2007. We observed that for p24, thereis no size dependent interaction with p24, however; we did see anincrease in gp41 binding in PS-ON randomers larger than 20 bases versusthose less than 20 bases. This suggests when PS-ON randomer lengthincreases above 20 bases, multiple copies of gp41 can bind to individualrandomers, increasing their polarization.

This is a significant observation as it demonstrates the potential oflarger ONs to sequester structural proteins during viral synthesis andlimit their availability for the formation of new virions.

High Affinity Oligonucleotides

Another approach is a method to enrich or purify antiviral ON(s) havinga higher affinity for viral components, such as viral proteins, than theaverage affinity of the ONs in a starting pool of ONs. The method willthus provide one or more non-sequence complementary ON(s) that willexhibit increased affinity to one or more viral components, e.g., havinga three-dimensional shape contributing to such elevated bindingaffinity. The rationale is that while ON(s) will act as linear moleculesin binding with viral components, they can also fold into a3-dimensional shape that can enhance the interaction with such viralcomponents. Without being limited to the specific technique, highaffinity ONs can be purified or enriched in the following ways.

One method for purifying a high affinity ON, or a plurality of highaffinity ONs, involves using a stationary phase medium with bound viralprotein(s) as an affinity matrix to bind ONs, which can then be elutedunder increasingly stringent conditions (e.g., increasing concentrationof salt or other chaotropic agent, and/or increasing temperture and/orchanges in pH). Such a method can, for example, be carried out by:

-   -   (a) loading a pool of ONs onto an exchange column having a viral        protein or several viral proteins or a viral lysate bound to a        stationary phase;    -   (b) displacing (eluting) bound ONs from the column, e.g., by        using a displacer solution such as an increasing salt solution;    -   (c) collecting fractions of eluted ONs at different salt        concentration;    -   (d) cloning and sequencing eluted ONs from different fractions,        more preferably from a fraction(s) at high salt concentration,        such that the ONs eluted at the high salt concentration have a        greater binding affinity with the viral protein(s); and    -   (e) Testing the activity of sequenced ON(s) in assays such        binding and/or viral inhibiton assay, e.g., a fluorescence        polarization-binding assay as decribed herein and/or in a        cellular viral inhibition assay and/or in an animal viral        inhibition assay.

In a second example, a method derived and modified from the SELEXmethodology (Morris et al (1998) Biochemistry 95:2902-2907) can be usedfor purifying the high affinity ON. One implementation of such a methodcan be performed as:

-   -   (a) providing a starting ON pool material, for example, a        collection of synthetic random ONs containing a high number of        sequences, e.g., one hundred trillion (10¹⁴) to ten quadrillion        (10¹⁶) different sequences. Each ON molecule contains a segment        of random sequence flanked by primer-binding sequences at each        end to facilitate polymerase chain reaction (PCR). Because the        nucleotide sequences of essentially all of the molecules are        unique, an enormous number of structures are sampled in the        population. These structures determine each molecule's        biochemical properties, such as the ability to bind a given        viral target molecule;    -   (b) contacting ONs with a viral protein or several viral        proteins or a viral lysate;    -   (c) selecting ONs that bind to viral protein(s), using a        partition technique(s) that can partition bound and unbound ONs,        such as native gel shifts and nitrocellulose filtration. Either        of these methods physically separates the bound species from the        unbound species, allowing preferential recovery of those        sequences that bind best. Also, to select ON (s) that bind to a        small protein, it is desirable to attach the target to a solid        support and use that support as an affinity purification matrix.        Those molecules that are not bound get washed off and the bound        ones are eluted with free target, again physically separating        bound and unbound species;    -   (d) amplifying the eluted binding ON(s), e.g., by using PCR        using primers hybridizing with both flanking sequences of ONs;    -   (e) steps (b) (c) and (d) can be performed multiple times (i.e.,        multiple cycles or rounds of enrichment and amplification) in        order to preferentially recover ONs that display the highest        binding affinity to viral protein(s). After several cycles of        enrichment and amplification, the population is dominated by        sequences that display the desired biochemical property;    -   (f) cloning and sequencing one or more ONs selected from from an        enrichment cycle, e.g., the last such cycle; and    -   (g) testing the binding and/or activity of sequenced ON(s) in        assays, e.g., in a fluorescence polarization binding assay as        decribed herein and/or in a cellular viral inhibition assay        and/or in an animal viral inhibition assay.

Another approach is to apply a modification of a split synthesismethodology to create one-bead one-PS-ON and one-bead one-PS2-ONlibraries as described in Yang et al (2002) Nucl. Acids Res.30(e132):1-8. Binding and selection of specific beads to viral proteinscan be done. Sequencing both the nucleic acid bases and the positions ofany thioate/dithioate linkages can be carried out by using a PCR-basedidentification tag of the selected beads. This approach can allow forthe rapid and convenient identification of PS-ONs or PS2-ONs that bindto viral proteins and that exhibit potent antiviral properties.

Once the specific sequences that bind to the viral proteins with highaffinity are determined (e.g., by amplification and sequencing ofindividual sequences), one or more such high affinity sequences can beselected and synthesized (e.g., by either chemical or enzymaticsynthesis) to provide a preparation of high affinity ON(s), which can bemodified to improve their activity, including improving theirpharmacokinetic properties. Such high affinity ONs can be used in thepresent invention.

Prion Diseases

Another approach is used in an alternative embodiment of the presentinvention for the treatment, the control of the progression, or theprevention of prion disease. This fatal neurodegenerative disease isinfectious and can affect both humans and animals. Structural changes inthe cellular prion protein, PrPC to its scrapie isoform, PrPSC, areconsidered to be the obligatory step in the occurrence and propagationof the prion disease. Amyloid polymers are associated withneuropathology of the prion disease.

The incubation of a prion protein fragment and double stranded nucleicacid results in the formation of amyloid fibres (Nandi et al (2002), JMol Biol 322: 153-161). ONs having affinity to proteins such asphosphorothioates are used to compete or inhibit the interaction ofdouble stranded nucleic acid with the PrPC and consequently stop theformation of the amyloid polymers. Such ONs of different sizes anddifferent compositions can be used in an appropriate delivery form totreat patients suffering from prion diseases or for prophylaxis in highrisk situations. Such interfering ONs can be identified by measuringfolding changes of amyloid polymerase as described by Nandi et al.(supra) in the presence of test ONs.

Putative Viral Etiologies

Another approach is used in another embodiment of the present inventionfor the treatment or prevention of diseases or conditions with putativeviral etiologies as described without limitation in the followingexamples. Viruses are putative causal agents in diseases and conditionsthat are not related to a primary viral infection. For example,arthritis is associated with HCV (Olivieri et al. (2003) Rheum Dis ClinNorth Am 29:111-122), Parvovirus B19, HIV, HSV, CMV, EBV, and VZV (Stahlet al. (2000) Clin Rheumatol 19:281-286). Other viruses have also beenidentified as playing a role in different diseases. For example,influenza A in Parkinson's disease (Takahashi et al. (1999), Jpn JInfect Dis 52:89-98), Coronavirus, EBV and other viruses in MultipleSclerosis (Talbot et al (2001) Curr Top Microbiol Immunol 253:247-71);EBV, CMV and HSV-6 in Chronic Fatigue Syndrome (Lerner et al. (2002)Drugs Today 38:549-561); and paramyxoviruses in asthma (Walter et al(2002) J Clin Invest 110:165-175) and in Paget's disease; and HBV, HSV,and influrenza in Guillain-Barre Syndrome.

Because of these etiologies, inhibition of the relevant virus using thepresent invention can delay, slow, or prevent development of thecorresponding disease or condition, or at least some symptoms of thatdisease.

Oligonucleotide Modifications and Synthesis

As indicated in the Summary above, modified oligonucleotides are usefulin this invention. Such modified oligonucleotides include, for example,oligonucleotides containing modified backbones or non-naturalinternucleoside linkages. Oligonucleotides having modified backbonesinclude those that retain a phosphorus atom in the backbone and thosethat do not have a phosphorus atom in the backbone.

Such modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates, carboranyl phosphate andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Oligonucleotides having inverted polarity typically include a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

Preparation of oligonucleotides with phosphorus-containing linkages asindicated above are described, for example, in U.S. Pat. Nos. 3,687,808;4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599;5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, each of whichis incorporated by reference herein in its entirety.

Some exemplary modified oligonucleotide backbones that do not include aphosphodiester linkage have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, 0, S and CH₂ component parts. Particularlyadvantageous are backbone linkages that include one or more chargedmoieties. Examples of U.S. patents describing the preparation of thepreceding oligonucleotides include U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437;5,792,608; 5,646,269 and 5,677,439, each of which is incorporated byreference herein in its entirety.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. For example, such oligonucleotides can include one of thefollowing 2′-modifications: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N— alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl, or 2′-O—(O-carboran-1-yl)methyl.Particular examples are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)˜OCH₃,O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to 10. Otherexemplary oligonucleotides include one of the following2′-modifications: C₁ to C₁₀ lower alkyl, substituted lower alkyl,alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃,OCN, Cl, Br, CN, CF₃.OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide. Examples include 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group;2′-dimethy-laminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE; and 2′-dimethylaminoethoxyethoxy (also known as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include Locked Nucleic Acids (LNAs) in which the2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugarring thereby forming a bicyclic sugar moiety. The linkage can be amethelyne (—CH₂—)˜ group bridging the 2′ oxygen atom and the 4′ carbonatom wherein n is 1 or 2. LNAs and preparation thereof are described inWO 98/39352 and WO 99/14226, which are incorporated herein by referencein their entireties.

Other modifications include sulfur-nitrogen bridge modifications, suchas locked nucleic acid as described in Orum et al. (2001) Curr. Opin.Mol. Ther. 3:239-243.

Other modifications include 2′-methoxy (2′-O—CH₃), 2′-methoxyethyl(2-O—CH₂—CH₃ ), 2′-ethyl, 2′-ethoxy, 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂),2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro(2′-F).

The 2′-modification may be in the arabino (up) position or ribo (down)position. Similar modifications may also be made at other positions onthe oligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of the 5′ terminal nucleotide. Oligonucleotides may also havesugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Exemplary U.S. patents describing the preparationof such modified sugar structures include, for example, U.S. Pat. Nos.4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; 5,792,747; and 5,700,920, each of which is incorporated byreference herein in its entirety.

Still other modifications include an ON concatemer consisting ofmultiple oligonucleotide sequences joined by a linker(s). The linkermay, for example, consist of modified nucleotides or non-nucleotideunits. In some embodiments, the linker provides flexibility to the ONconcatemer. Use of such ON concatemers can provide a facile method tosynthesize a final molecule, by joining smaller oligonucleotide buildingblocks to obtain the desired length. For example, a 12 carbon linker(C12 phosphoramidite) can be used to join two or more ON concatemers andprovide length, stability, and flexibility.

As used herein, “unmodified” or “natural” bases (nucleobases) includethe purine bases adenine (A) and guanine (G), and the pyrimidine basesthymine (T), cytosine (C) and uracil (U). Oligonucleotides may alsoinclude base modifications or substitutions. Modified bases includeother synthetic and naturally-occurring bases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl(-C≡C—CH₃) uracil and cytosine and other alkynylderivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine,5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additionalmodified bases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine(2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases mayalso include those in which the purine or pyrimidine base is replacedwith other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine,2-aminopyridine and 2-pyridone. Further nucleobases include thosedescribed in U.S. Pat. No. 3,687,808, those disclosed in The ConciseEncyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993.

Another modification includes phosphorodithioate linkages. Knowing thatphosphorodithioate ONs (PS2-ONs) and PS-ONs have a similar bindingaffinity to proteins (Tonkinson et al. (1994) Antisense Res. Dev. 4:269-278)(Cheng et al. (1997) J. Mol. Recogn. 10:101-107) and knowingthat a possible mechanism of action of ONs is binding to viral proteins,it could be desirable to include phosphorodithioate linkages on theantiviral ONs described in this invention.

Another approach to modify ONs is to produce stereodefined orstereo-enriched ONs as described in Yu at al (2000) Bioorg. Med. Chem.8:275-284 and in Inagawa et al. (2002) FEBS Lett. 25:48-52. ONs preparedby conventional methods consist of a mixture of diastereomers by virtueof the asymmetry around the phosphorus atom involved in theinternucleotide linkage. This may affect the stability of the bindingbetween ONs and viral components such as viral proteins. Previous datashowed that protein binding is significantly stereo-dependent (Yu etal.). Thus, using stereodefined or stereo-enriched ONs could improvetheir protein binding properties and improve their antiviral efficacy.

The incorporation of modifications such as those described above can beutilized in many different incorporation patterns and levels. That is, aparticular modification need not be included at each nucleotide orlinkage in an oligonucleotide, and different modifications can beutilized in combination in a single oligonucleotide, or even in a singlenucleotide.

As examples and in accordance with the description above, modifiedoligonucleotides containing phosphorothioate or dithioate linkages mayalso contain one or more substituted sugar moieties particularlymodifications at the sugar moieties including, without restriction,2′-ethyl, 2′-ethoxy, 2′-methoxy, 2′-aminopropoxy, 2′-allyl, 2′-fluoro,2′-pentyl, 2′-propyl, 2′-dimethylaminooxyethoxy, and2′-dimethylaminoethoxyethoxy. The 2′-modification may be in the arabino(up) position or ribo (down) position. A preferred 2′-arabinomodification is 2′-fluoro. Similar modifications may also be made atother positions on the oligonucleotide, particularly the 3′ position ofthe sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Moreover ONs may have astructure of or comprise a portion consisting of glycol nucleic acid(GNA) with an acyclic propylene glycol phosphodiester backbone (Zhang L,et al (2005) J. Am. Chem. Soc. 127(12):4174-5). Such GNA may comprisephosphorothioate linkages and may comprise only pyrimidine bases.

Oligonucleotide Synthesis

The present oligonucleotides can by synthesized using methods known inthe art. For example, unsubstituted and substituted phosphodiester (P═O)oligonucleotides can be synthesized on an automated DNA synthesizer(e.g., Applied Biosystems model 380B) using standard phosphoramiditechemistry with oxidation by iodine. Phosphorothioates (P═S) can besynthesized as for the phosphodiester oligonucleotides except thestandard oxidation bottle can be replaced by 0.2 M solution of311-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for thestep-wise thioation of the phosphite linkages. The thioation wait stepcan be increased to 68 sec, followed by the capping step. After cleavagefrom the CPG column and deblocking in concentrated ammonium hydroxide at55° C. (18 h), the oligonucleotides can be purified by precipitatingtwice with 2.5 volumes of ethanol from a 0.5 M NaCl solution.

Phosphinate oligonucleotides can be prepared as described in U.S. Pat.No. 5,508,270; alkyl phosphonate oligonucleotides can be prepared asdescribed in U.S. Pat. No. 4,469,863; 3′-Deoxy-3′-methylene phosphonateoligonucleotides can be prepared as described in U.S. Pat. Nos.5,610,289 and 5,625,050; phosphoramidite oligonucleotides can beprepared as described in U.S. Pat. No. 5,256,775 and U.S. Pat. No.5,366,878; alkylphosphonothioate oligonucleotides can be prepared asdescribed in published PCT applications PCT/US94/00902 andPCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively);3′-Deoxy-3′-amino phosphoramidate oligonucleotides can be prepared asdescribed in U.S. Pat. No. 5,476,925; Phosphotriester oligonucleotidescan be prepared as described in U.S. Pat. No. 5,023,243; boranophosphateoligonucleotides can be prepared as described in U.S. Pat. Nos.5,130,302 and 5,177,198; methylenemethylimino linked oligonucleotides,also identified as MMI linked oligonucleotides,methylenedimethyl-hydrazo linked oligonucleotides, also identified asMDII linked oligonucleotides, and methylenecarbonylamino linkedoligonucleotides, also identified as amide-3 linked oligonucleotides,and methyleneaminocarbonyl linked oligo-nucleotides, also identified asamide-4 linked oligonucleo-sides, as well as mixed backbone compoundshaving, for instance, alternating MMI and P═O or P═S linkages can beprepared as described in U.S. Pat. Nos. 5,378, 825, 5,386,023,5,489,677, 5,602,240 and 5,610,289; formacetal and thioformacetal linkedoligonucleotides can be prepared as described in U.S. Pat. Nos.5,264,562 and 5,264,564; and ethylene oxide linked oligonucleotides canbe prepared as described in U.S. Pat. No. 5,223,618. Each of the citedpatents and patent applications is incorporated by reference herein inits entirety.

Oligonucleotide Formulations and Pharmaceutical Compositions

The present oligonucleotides can be prepared in an oligonucleotideformulation or pharmaceutical composition. Thus, the presentoligonucleotides may also be admixed, encapsulated, conjugated orotherwise associated with other molecules, molecule structures ormixtures of compounds, as for example, liposomes, receptor targetedmolecules, oral, rectal, topical or other formulations, for assisting inuptake, distribution and/or absorption. Exemplary U.S. patents thatdescribe the preparation of such uptake, distribution and/or absorptionassisting formulations include, for example, U.S. Pat. Nos. 5,108,921;5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932;5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921;5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016;5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259;5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which isincorporated herein by reference in its entirety.

The oligonucleotides, formulations, and compositions of the inventioninclude any pharmaceutically acceptable salts, esters, or salts of suchesters, or any other compound which, upon administration to an animalincluding a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof. Accordingly, forexample, the disclosure is also drawn to prodrugs and pharmaceuticallyacceptable salts of the compounds of the invention, pharmaceuticallyacceptable salts of such prodrugs, and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular embodiments, prodrug versionsof the present oligonucleotides are prepared as SATE[(S-acetyl-2-thioethyl)phosphate] derivatives according to the methodsdisclosed in Gosselin et al., WO 93/24510 and in Imbach et al., WO94/26764 and U.S. Pat. No. 5,770,713, which are hereby incorporated byreference in their entireties.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the present compounds: i.e.,salts that retain the desired biological activity of the parent compoundand do not impart undesired toxicological effects thereto. Many suchpharmaceutically acceptable salts are known and can be used in thepresent invention.

For oligonucleotides, useful examples of pharmaceutically acceptablesalts include but are not limited to salts formed with cations such assodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; acid addition salts formed with inorganicacids, for example hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, and the like; and salts formed fromelemental anions such as chlorine, bromine, and iodine.

The present invention also includes pharmaceutical compositions andformulations which contain the antiviral oligonucleotides of theinvention. Such pharmaceutical compositions may be administered in anumber of ways depending upon whether local or systemic treatment isdesired and upon the area to be treated. For example, administration maybe topical (including ophthalmic and to mucous membranes includingvaginal and rectal delivery); pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal; intranasal; epidermal and transdermal; oral; orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Preferred topical formulations include those inwhich the oligonucleotides of the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Preferredlipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidylglycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters include but are not limited arachidonic acid, oleicacid, eicosanoic acid, laurie acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Exemplary surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Exemplary bileacids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenedeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate. Exemplaryfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also preferred are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly preferred combination is the sodium salt oflauric acid, capric acid and UDCA. Further exemplary penetrationenhancers include polyoxyethylene-9-lauryl ether,polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may bedelivered orally in granular form including sprayed dried particles, orcomplexed to form micro or nanoparticles. Oligonucleotide complexingagents include poly-amino acids; polyimines; polyacrytates;polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationizedgelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) andstarches; polyalkylcyanoacrylates; DEAE-derivatized polyimines,pollulans, celluloses, and starches. Particularly advantageouscomplexing agents include chitosan, N-trimethytchitosan, poly-L-lysine,polyhistidine, polyorithine, polyspermines, protamine,polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE),polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate),poly(ethylcyanoacrylate), poly(butylcyanoacrylatc),poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate),DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin andDEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lacticacid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, andpolyethyleneglycol (PEG).

Compositions for vaginal delivery can be in various forms, including forexample, a gel, cream, tablet, pill, capsule, suppository, film, or anyother pharmaceutically acceptable form that adheres to the mucosa anddoes not wash away easily. A large variety of different formulations forvaginal delivery are further described in the art, for example in U.S.Pat. Nos. 4,615,697 and 6,699,494, which are incorporated herein byreference in their entireties.

Additionally, additives (such as those described in the U.S. Pat. No.4,615,697 patent) may be combined in the formulation for maximum ordesired efficacy of the delivery system or for the comfort of thepatient. Such additives include, for example, lubricants, plasticizingagents, preservatives, gel formers, tablet formers, pill formers,suppository formers, film formers, cream formers, disintegrating agents,coatings, binders, vehicles, coloring agents, taste and/or odorcontrolling agents, humectants, viscosity controlling agents,pH-adjusting agents, and similar agents.

In certain embodiments, a composition can include a cross-linkedpolycarboxylic acid polymer formulation, generally described in U.S.Pat. No. 4,615,697. In general, in such embodiments at least eightypercent of the monomers of the polymer in such a formulation shouldcontain at least one carboxyl functionality. The cross-linking agentshould be present at such an amount as to provide enough bioadhesion toallow the system to remain attached to the target epithelial surfacesfor a sufficient time to allow the desired dosing to take place.

For vaginal administration, such a formulation remains attached to theepithelial surfaces for a period of at least about twenty-four toforty-eight hours. Such results may be measured clinically over variousperiods of time, by testing samples from the vagina for pH reduction dueto the continued presence of the polymer. This preferred level ofbioadhesion is usually attained when the cross-linking agent is presentat about 0.1 to 6.0 weight percent of the polymer, with about 1.0 to 2.0weight percent being most preferred, as long as the appropriate level ofbioadhesion results. Bioadhesion can also be measured by commerciallyavailable surface tensiometers utilized to measure adhesive strength.

The polymer formulation can be adjusted to control the release rate byvarying the amount of cross-linking agent in the polymer. Suitablecross-linking agents include divinyl glycol, divinylbenzene,N,N-diallylacrylamide, 3,4-dihydroxy-1,5-hexadiene,2,5-dimethyl-1,5-hexadiene and similar agents.

A preferred polymer for use in such a formulation is Polycarbophil,U.S.P., which is commercially available from B. F. Goodrich SpecialityPolymers of Cleveland, Ohio under the trade name NOVEON.RTM.-AA1. TheUnited States Pharmacopeia, 1995 edition, United States PharmacopeialConvention, Inc., Rockville, Md., at pages 1240-41, indicates thatpolycarbophil is a polyacrylic acid, cross-linked with divinyl glycol.

Other useful bioadhesive polymers that may be used in such a drugdelivery system formulation are mentioned in the U.S. Pat. No. 4,615,697patent. For example, these include polyacrylic acid polymerscross-linked with, for example, 3,4-dihydroxy-1,5-hexadiene, andpolymethacrylic acid polymers cross-linked with, for example, divinylbenzene. Typically, these polymers would not be used in their salt form,because this would decrease their bioadhesive capability. Suchbioadhesive polymers may be prepared by conventional free radicalpolymerization techniques utilizing initiators such as benzoyl peroxide,azobisisobutyronitrile, and the like. Exemplary preparations of usefulbioadhesives are provided in the U.S. Pat. No. 4,615,697 patent.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shakingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

The formulations and compositions of the present invention may beprepared and formulated as emulsions. Emulsions are typicallyheterogenous systems of one liquid dispersed in another in the form ofdroplets usually exceeding 0.1 μm in diameter. (Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (lids.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et at., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising of two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: non-ionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong inter-facial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid, Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of reasons of ease of formulation, efficacyfrom an absorption and bioavailabiity standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically micro-emulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML31O), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschet, Met/i. Find.Exp. Clin. PharmacoL, 1993, 13, 205). Micro-emulsions afford advantagesof improved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset at., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Set, 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucteotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92).

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles offerspecificity and extended duration of action for drug delivery. Thus, asused herein, the term “liposome” refers to a vesicle composed ofamphiphilic lipids arranged in a spherical bilayer or bilayers, i.e.,liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion typically contains the composition to be delivered. Inorder to cross intact mammalian skin, lipid vesicles must pass through aseries of fine pores, each with a diameter less than 50 nm, under theinfluence of a suitable transdermal gradient. Therefore, it is desirableto use a liposome which is highly deformable and able to pass throughsuch fine pores. Additional factors for liposomes include the lipidsurface charge, and the aqueous volume of the liposomes.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245).

For topical administration, there is evidence that liposomes presentseveral advantages over other formulations. Such advantages includereduced side-effects related to high systemic absorption of theadministered drug, increased accumulation of the administered drug atthe desired target, and the ability to administer a wide variety ofdrugs, both hydrophilic and hydrophobic, into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin, generally resulting intargeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etat., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs. TheDNA is thus entrapped in the aqueous interior of these liposomes.pH-sensitive liposomes have been used, for example, to deliver DNAencoding the thymidine kinase gene to cell monolayers in culture (Zhouet al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et at., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasone™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et at. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome include one ormore glycolipids, such as monosialoganglioside G_(M1), or is derivatizedwith one or more hydrophilic polymers, such as a polyethylene glycol(PEG) moiety. Without being bound by any particular theory, it isbelieved that for sterically stabilized liposomes containinggangliosides, sphingomyelin, or PEG-derivatized lipids, the increase incirculation half-life of these sterically stabilized liposomes is due toa reduced uptake into cells of the reticuloendothelial system (RES)(Allen et at., FEBS Lett., 1987, 223, 42; Wu et al., Cancer Research,1993, 53, 3765).

Various liposomes that include one or more glycolipids have beenreported in Papahadjopoulos et al., Ann. N.Y. Acad. Sci., 1987, 507, 64(monosiatoganglioside G_(M1), galactocerebroside sulfate andphosphatidylinositol); Gabizon et at., Proc. Natl. Acad. Sci. USA.,1988, 85, 6949; Allen et al., U.S. Pat. No. 4,837,028 and InternationalApplication Publication WO 88/04924 (sphingomyelin and the gangliosideG_(M1) or a galactocerebroside sulfate ester); Webb et al., U.S. Pat.No. 5,543,152 (sphingomyelin); Lim et al., WO 97/13499(1,2-sn-dimyristoylphosphatidylcholine).

Liposomes that include lipids derivatized with one or more hydrophilicpolymers, and methods of preparation are described, for example, inSunamoto et al., Bull. Chem. Soc. Jpn., 1980, 53, 2778 (a nonionicdetergent, 2C₁₂15G, that contains a PEG moiety); Illum et al., FEBSLett, 1984, 167, 79 (hydrophilic coating of polystyrene particles withpolymeric glycols); Sears, U.S. Pat. Nos. 4,426,330 and 4,534,899(synthetic phospholipids modified by the attachment of carboxylic groupsof polyalkylene glycols (e.g., PEG)); Klibanov et al., FEBS Lett., 1990,268, 235 (phosphatidylethanolamine (PE) derivatized with PEG or PEGstearate); Blume et al., Biochimica et Biophysica Acta, 1990, 1029, 91(PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from thecombination of distearoylphosphatidylethanolamine (DSPE) and PEG);Fisher, European Patent No. EP 0 445 131 B1 and WO 90/04384 (covalentlybound PEG moieties on liposome external surface); Woodle et al., U.S.Pat. Nos. 5,013,556 and 5,356,633, and Martin et al., U.S. Pat. No.5,213,804 and European Patent No. EP 0 496 813 B1 (liposome compositionscontaining 1-20 mole percent of PE derivatized with PEG); Martin et al.,WO 91/05545 and U.S. Pat. No. 5,225,212 and in Zalipsky et al., WO94/20073 (liposomes containing a number of other lipid-polymerconjugates); Choi et al., WO 96/10391 (liposomes that includePEG-modified ceramide lipids); Miyazaki et al., U.S. Pat. No. 5,540,935,and Tagawa et al., U.S. Pat. No. 5,556,948 (PEG-containing liposomesthat can be further derivatized with functional moieties on theirsurfaces).

Liposomes that include nucleic acids have been described, for example,in Thierry et al., WO 96/40062 (methods for encapsulating high molecularweight nucleic acids in liposomes); Tagawa et al., U.S. Pat. No.5,264,221 (protein-bonded liposomes containing RNA); Rahman et al., U.S.Pat. No. 5,665,710 (methods of encapsulating oligodeoxynucleotides inliposomes); Love et al., WO 97/04787 (liposomes that include antisenseoligonucleotides).

Another type of liposome, transfersomes are highly deformable lipidaggregates which are attractive for drug delivery vehicles. (Cevc etal., 1998, Biochim Biophys Acta. 1368(2):201-15.) Transfersomes may bedescribed as lipid droplets which are so highly deformable that they canpenetrate through pores which are smaller than the droplet.Transfersomes are adaptable to the environment in which they are used,for example, they are shape adaptive, self-repairing, frequently reachtheir targets without fragmenting, and often self-loading. Transfersomescan be made, for example, by adding surface edge-activators, usuallysurfactants, to a standard liposomal composition.

Surfactants

Surfactants are widely used in formulations such as emulsions (includingmicroemulsions) and liposomes. The most common way of classifying andranking the properties of the many different types of surfactants, bothnatural and synthetic, is by the use of the hydrophile/lipophile balance(HLB). The nature of the hydrophilic group (also known as the “head”)provides the most useful means for categorizing the differentsurfactants used in formulations (Rieger, in Pharmaceutical DosageForms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants are widely used inpharmaceutical and cosmetic products and are usable over a wide range ofpH values, and with typical HLB values from 2 to about 18 depending onstructure. Nonionic surfactants include nonionic esters such as ethyleneglycol esters, propylene glycol esters, glyceryl esters, polyglycerylesters, sorbitan esters, sucrose esters, and ethoxylated esters; andnonionic alkanolamides and ethers such as fatty alcohol ethoxylates,propoxylated alcohols, and ethoxylated/propoxylated block polymers arealso included in this class. The polyoxyethylene surfactants are themost commonly used members of the nonionic surfactant class.

Surfactant molecules that carry a negative charge when dissolved ordispersed in water are classified as anionic. Anionic surfactantsinclude carboxylates such as soaps, acyl lactylates, acyl amides ofamino acids, esters of sulfuric acid such as alkyl sulfates andethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates,acyl isothionates, acyl laurates and sulfosuccinates, and phosphates.The alkyl sulfates and soaps are the most commonly used anionicsurfactants.

Surfactant molecules that carry a positive charge when dissolved ordispersed in water are classified as cationic. Cationic surfactantsinclude quaternary ammonium salts and ethoxylated amines, with thequaternary ammonium salts used most often.

Surfactant molecules that can carry either a positive or negative chargeare classified as amphoteric. Amphoteric surfactants include acrylicacid derivatives, substituted alkylamides, N-alkylbetaines andphosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed in Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In some embodiments, penetration enhancers are used in or with acomposition to increase the delivery of nucleic acids, particularlyoligonucleotides, to the skin or across mucous membranes of animals.Most drugs are present in solution in both ionized and nonionized forms.However, usually only lipid soluble or lipophilic drugs readily crosscell membranes. It has been discovered that even non-lipophilic drugsmay cross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating nonsurfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classesof penetration enhancers is described below in greater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. These penetration enhancers include, for example, sodiumlauryl sulfate, polyoxyethylene-9-lauryl ether andpolyoxyethylene-20-cetyl ether) (Lee et at., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252), each of which is incorporated herein by reference in itsentirety.

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and diglycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654),each of which is incorporated herein by reference in its entirety.

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: In the present context, chelating agents can beregarded as compounds that remove metallic ions from solution by formingcomplexes therewith, with the result that absorption of oligonucleotidesthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Without limitation, chelating agents include disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones(enamines) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds are compounds that do notdemonstrate significant chelating agent or surfactant activity, butstill enhance absorption of oligonucleotides through the alimentarymucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33). Examples of such penetration enhancers includeunsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and nonsteroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions andformulations of the present invention. For example, cationic lipids,such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationicglycerol derivatives, and polycationic molecules, such as polylysine(Lollo et al., PCT Application WO 97/30731), are also known to enhancethe cellular uptake of oligonucleotides.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, often with an excess of the latter substance, can result in asubstantial reduction of the amount of nucleic acid recovered in theliver, kidney or other extracirculatory reservoirs. For example, therecovery of a partially phosphorothioate oligonucleotide in hepatictissue can be reduced when it is coadministered with polyinosinic acid,dextran sulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995,5, 115-121; Takakura et al., Antisense & NucLAcid Drug Dev., 1996, 6, 177-183), each of which is incorporated hereinby reference in its entirety.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal, and is typically liquid or solid. Apharmaceutical carrier is generally selected to provide for the desiredbulk, consistency, etc., when combined with a nucleic acid and the othercomponents of a given pharmaceutical composition, in view of theintended administration mode. Typical pharmaceutical carriers include,but are not limited to, binding agents (e.g., pregelatinized maizestarch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.);fillers (e.g., lactose and other sugars, microcrystalline cellulose,pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates orcalcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate,talc, silica, colloidal silicon dioxide, stearic acid, metallicstearates, hydrogenated vegetable oils, corn starch, polyethyleneglycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g.,starch, sodium starch glycotate, etc.); and wetting agents (e.g., sodiumlauryl sulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Other Pharmaceutical Composition Components

The present compositions may additionally contain other componentsconventionally found in pharmaceutical compositions, at theirart-established usage levels. Thus, for example, the compositions maycontain additional, compatible, pharmaceutically-active materials suchas, for example, antipruritics, astringents, local anesthetics oranti-inflammatory agents, or may contain additional materials useful inphysically formulating various dosage forms of the compositions of thepresent invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions of thepresent invention. The formulations can be sterilized and, if desired,mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, colorings, flavorings and/or aromatic substances andthe like which do not deleteriously interact with the nucleic acid(s) ofthe formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran, and/or stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antiviral oligonucleotides and (b) one ormore other chemotherapeutic agents which function by a differentmechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethytmetamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin, and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-EU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toRibavirin, cidofovir, vidarabine, acyclovir and ganciclovir, may also becombined in compositions of the invention. See, generally, The MerckManual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987,Rahway, N.J., pages 2499-2506 and 46-49, respectively). Othernon-oligonucleotide chemotherapeutic agents are also within the scope ofthis invention. Two or more combined compounds may be used together orsequentially.

EXAMPLES Example 1 Herpes Simplex Virus

Herpes simplex virus (HSV) affects a significant proportion of the humanpopulation. It was found in the present invention that random ONs or ONrandomers inhibited the infectivity of viruses such as HSV. Usingcellular HSV replication assays in VERO cells (susceptible to HSV-1(strain KOS) and HSV-2 (strain MS2) infection) it was found that asingle stranded PS-ON complementary to the HSV origin of replicationinhibited replication of HSV-1 and HSV-2. Surprisingly, control PS-ONscomplementary to human (343 ARS) and plasmid (pBR322/pUC) origins alsoinhibited viral infectivity. Experiments with random sequence PS-ONs andPS-ON randomers demonstrated that inhibition of viral infectionincreased with increasing ON size. These data show that ONs are potentantiviral agents useful for therapeutic treatment of viral infection.

The inventors have theorized that a potential mechanism for blocking thespread of viruses such as HHVs was to prevent the replication of itsDNA. With this in mind, phosphorothioate oligonucleotides (ONs)complementary to the origin of replication of HSV1 and HSV2 wereintroduced into infected cells. These ONs would cause DNA triplexformation at the viral origin of replication, blocking the associationof necessary trans-acting factors and viral DNA replication. Surprisingresults are presented herein of these experiments which show that, in anexperimental paradigm, the potency of ONs in inhibiting viral infectionincreases as their size (length) increases.

Inhibition of HSV-1

The ability of PS-ONs to inhibit HSV-1 is measured in a plaque reductionassay (PRA). Immortalized African Green Monkey kidney (VERO) cells arecultured at 37° C. and 5% CO₂ in MEM (minimal essential medium) plus 10%fetal calf serum supplemented with gentamycin, vancomycin andamphoterecin B. Cells are seeded in 12 well plates at a density whichyields a confluent monolayer of cells after 4 days of growth. Uponreaching confluency, the media is changed to contain only 5% serum plussupplements as described above and cells are then exposed to HSV-1(strain KOS, approximately 40-60 PFU total) in the presence of the testcompound for 90 minutes. After viral exposure, the media is replacedwith new “overlay” media containing 5% serum, 1% human immunoglobulins,supplements as described above and the test compound. Plaque counting isperformed 3-4 days post infection following formalin fixation and cresylviolet staining of infected cultures.

All ONs (except where noted otherwise) were synthesized at theUniversity of Calgary Core DNA Services lab. ONs (see table 21) areprepared on a 1 or 15 micromol synthesis scale, deprotected and desaltedon a 50 cm Sephadex G-25 column. The resulting ONs are analyzed by UVshadowing gel electrophoresis and are determined to contain ˜95% of thefull length, n-1 and n-2 oligo and up to 5% of shorter oligo species(these are assumed to have random deletions). For random oligosynthesis, adenine, guanosine, cytosine and thymidine amidites are mixedtogether in equimolar quantities to maximize the randomness ofincorporation at each position of the ONs during synthesis.

To test if PS-ONs could inhibit HSV-1, REP 1001, 2001 and 3007 aretested in the HSV-1 PRA. It would have been expected that only REP 2001will show any activity as this PS-ON is directed against the origin ofreplication in HSV (the other two are directed against replicationorigins in humans and plasmids). However all three PS-ONs showedanti-HSV-1 activity. The testing was carried out in a plaque reductionassay conducted in VERO cells using HSV-1 (strain KOS). Infected cellswere treated with increasing concentrations of REP 1001, REP 2001, orREP 3007. IC₅₀ values calculated from linear regressions of the assayresults were 2.76, 0.77, and 5.33 micromolar respectively. Moreover, thepotentcy of the anti-HSV-1 effect was found to be dependent on the sizeof the oligo.

To confirm the size dependence and relative sequence independence ofPS-ONs on anti-HSV-1 activity, we tested PS-ONs that vary in size (REP2002, 2003, 2004, 2005 and 2006) along with the antiviral drugAcyclovir. These PS-ONs are rendered inert with respect to sequencespecific effects by synthesizing each base as a “wobble” (N) so thateach PS-ON actually represents a population of different randomsequences with the same size; these PS-ONs are termed “randomers”.Plaque reduction assay was conducted in VERO cells using HSV-1 (strainKOS). Infected cells are treated with increasing concentrations of REP2001, REP 2002 or REP 3003, REP 2004, REP 2005, REP 2006, and Acyclovir.IC₅₀ values were calculated from linear regressions of assay data. Therelationship between PS-ON size and IC₅₀ against HSV-1 was determined byplotting the IC₅₀ values against the specific size of each PS-ON testedwhich showed anti-HSV-1 activity. The IC₅₀ for Acyclovir was used as areference to a clinical correlate. We found that oligos 10 bases orlower have no detectable anti-HSV-1 activity but as the size of thePS-ON increases above 10 bases, the potency also increases (IC₅₀decreases). We also noted that PS-ONs greater than 20 bases had IC₅₀values significantly lower than a clinically accepted anti-HSV-1 drug,acyclovir.

To better define the effective size range for PS-ON anti-HSV-1 activity,we tested PS-ON randomers covering a broader range of sizes from 10 to120 bases. Plaque reduction assay was conducted in VERO cells usingHSV-1 (strain KOS). A broad range of PS-ON randomer sizes were tested inincreasing concentrations; REP 2003, REP 2009, REP 2010, REP 2011, REP2012, REP 2004, REP 2006, REP 2007, and REP 2008. IC₅₀ values werecalculated from linear regressions. We discovered that oligos 12 basesand larger have detectable anti-HSV-1 activity and that the efficacyagainst HSV-1 also increases with increased PS-ON randomer length up toat least 120 bases. However, the increases in efficacy per base increasein size are smaller in PS-ON randomers greater than 40 bases.

To compare the efficacy of non-PS-ON randomers, a random sequence PS-ONand a HSV-1 specific sequence PS-ON, we tested these three types ofmodifications in ONs 10, 20 and 40 bases in size. Plaque reduction assaywas conducted in VERO cells using HSV-1 (strain KOS). Unmodified ONs,PS-ONs with a random sequence, and PS-ONs targeting the start codon ofHSV-1 IE110 were tested in increasing concentrations. The ONs were REP2013, REP 2014, REP 2015, REP 2016, REP 2017, REP 2018, REP 2019, REP2020, and REP 2021. IC₅₀ values were calculated from linear regressions.In this system, unmodified ON randomers have no detectable anti-HSV-1activity at tested sizes. Both random sequence and specific HSV-1sequence PS-ONs show size dependent anti-HSV-1 activity (no activity isobserved at 10 bases for either of these modifications. A comparison ofrandom sequence, specific HSV-1 sequence and randomer PS-ONs showed thatfor PS-ONs 20 bases in length, there is an enhancement of anti-HSV-1activity with the specific HSV-1 sequence but that at 40 bases inlength, all modifications, whether randomer, random sequence or specificHSV-1 sequence were equally efficacious against HSV-1.

To the best of our knowledge, this is the first time IC₅₀s for HSV-1 aslow as 0.059 μM and 0.043 μM are reported for PS-ONs.

Example 2 Inhibition of HSV-2

The ability of PS-ONs to inhibit HSV-2 is measured by PRA. ImmortalizedAfrican Green Monkey kidney (VERO) cells are cultured at 37° C. and 5%CO₂ in MEM plus 10% fetal calf serum supplemented with gentamycin,vancomycin and amphoterecin B. Cells are seeded in 12 well plates at adensity which yields a confluent monolayer of cells after 4 days ofgrowth. Upon reaching confluency, the media is changed to contain only5% serum plus supplements as described above and cells are then exposedto HSV-2 (strain MS2, approximately 40-60 PFU total) in the presence ofthe test compound for 90 minutes. After viral exposure, the media isreplaced with new “overlay” media containing 5% serum, 1% humanimmunoglobulins, supplements as described above and the test compound.Plaque counting is performed 3-4 days post infection following formalinfixation and cresyl violet staining of infected cultures.

To test if PS-ONs could inhibit HSV-2, REP 1001, 2001 and 3007 aretested in the HSV-2 PRA. Plaque reduction assay was conducted in humanfibroblast cells using HSV-2 (strain MS2), with infected cells treatedwith increasing concentrations of REP 1001, REP 2001, or REP 3007. IC₅₀values were calculated from linear regressions. If the inhibitoryactivity were due to an antisense or other sequence complementarymechanism, it would be expected that only REP 2001 would show anyactivity as this PS-ON is directed against the origin of replication inHSV-1/2 (the other two are directed against replication origins inhumans and plasmids respectively). However all three PS-ONs showedanti-HSV-2 activity. Moreover, the potency of the anti-HSV-2 effect isdependent on the size of the PS-ON and independent of the sequence.

To confirm the size dependence and sequence independence of PS-ONs onanti-HSV-2 activity, we tested PS-ONs that vary in size (REP 2001, 2002,2003, 2004, 2005 and 2006). These PS-ONs are rendered inert with respectto sequence specific effects by synthesizing each base as a “wobble” (N)so that each PS-ON actually represents a population of different randomsequences with the same size, these PS-ONs are termed “randomers”. Whenthese PS-ONs are tested in the HSV-2 PRA, we find that PS-ONs 10 basesor lower had no detectable anti-HSV-2 activity but as the size of thePS-ON increases above 10 bases, the potency also increases (IC₅₀decreases). We also noted that PS-ONs greater than 20 bases had IC₅₀values significantly lower than a clinically accepted anti-HSV-2 drug,acyclovir™.

To the best of our knowledge, this is the first time an IC₅₀ for HSV-2as low as 0.012 μM has been reported for a PS-ON.

To determine if non-specific sequence composition has an effect on ONantiviral activity, several PS-ONs of equivalent size but differing intheir sequence composition were tested for anti-HSV1 activity in theHSV-1 PRA. The PS-ONs tested were REP 2006 (N20), REP 2028 (G40), REP2029 (A40), REP 2030 (T40) and REP 2031 (C40). The IC₅₀ values generatedfrom the HSV-1 PRA show that REP 2006 (N40) was the most active of allsequences tested while REP 2029 (A40) was the least active. We also notethat, all the other PS-ONs were significantly less active than N40 withtheir rank in terms of efficacy being N40>C40>T40>A40>>G40.

We also tested the efficacy of different PS ONs having varying sequencecomposition with two different nucleotides. The PS-ON randomer (REP2006) was significantly more efficacious against HSV-1 than AC20 (REP2055), TC20 (REP 2056) or AG20 (REP 2057) with their efficacies rankedas follows: N40>AG>AC>TC. This data suggests that although theanti-viral effect is non-sequence complementary, certain non-specificsequence compositions (ie C40 and N40) have more potent anti-viralactivity. We suggest that this phenomenon can be explained by the factthat, while retaining intrinsic protein binding ability, sequences likeC40, A40, T40 and G40 bind fewer viral proteins with high affinity,probably due to some restrictive tertiary structure formed in thesesequences. On the other hand, due to the random nature of N40, itretains its ability to bind with high affinity to a broad range ofanti-viral proteins which contributes to its robust anti-viral activity.

Example 3 Inhibition of CMV

The ability of PS-ONs to inhibit CMV is measured in a plaque reductionassay (PRA). This assay is identical to the assay used for testinganti-HSV-1 and anti-HSV-2 except that CMV (strain AD169) is used as theviral innoculum and human fibroblasts were used as cellular host.

To test the size dependence and sequence independence of PS-ONs onanti-CMV activity, we tested PS-ON randomers that vary in size. Plaquereduction assay was conducted in VERO cells using CMV (strain AD169).Infected cells were treated with increasing concentrations of REP 2004(a) or REP 2006 (b). IC₅₀ values were calculated from linearregressions, and relationship between PS-ON size and IC₅₀ against CMVwas determined by plotting IC₅₀ values against the specific size of eachPS-ON tested. When these PS-ONs are tested in the CMV PRA, we find thatas the size of the PS-ON increases, the potency also increases (IC₅₀decreases).

To more clearly elucidate the effective size range for PS-ON anti-CMVactivity, we tested PS-ON randomers covering a broader range of sizesfrom 10 to 80 bases. We also included several clinically accepted smallmolecule CMV treatments (Gancyclovir, Foscarnet and Cidofovir) as wellas 2 versions of a marketed antisense treatment for CMV retinitis,(Vitravene™; commercially available and synthesized by the University ofCalgary). Plaque reduction assay was conducted in VERO cells using CMV(strain AD169). Three clinical CMV therapies were tested: Gancyclovir,Foscarnet, and Cidofovir. A broad range of PS-ON randomer sizes werealso tested in increasing concentrations; REP 2003, REP 2004, REP 2006,and REP 2007. Finally, REP 2036 (Vitravene) was tested as synthesized inhouse and as commercially available. IC₅₀ values were calculated fromlinear regressions. We discovered that while increased PS-ON randomersize leads to increased efficacy, this effect saturates at about 40bases. Moreover, the 20, 40 and 80 base PS-ON randomers are allsignificantly more efficacious than any of the small molecule treatmentstested. In addition, 40 and 80 base PS-ON randomers are more efficaciousthan Vitravene™.

To the best of our knowledge, this is the first time an IC₅₀ for CMV aslow as 0.067 μM has been reported for a PS-ON.

Example 4 Inhibition of HIV-1

The ability of PS-ON randomers to inhibit HIV-1 is measured by twodifferent assays:

Cytopathic Effect (CPE)

Cytopathic effect is monitored using MTT dye to report the extent ofcellular metabolism. Immortalized human lymphocyte (MT4) cells arecultured at 37° C. and 5% CO₂ in MEM plus 10% fetal calf serumsupplemented with antibiotics. Cells are seeded in 96 well plates inmedia containing the appropriate test compound and incubated for 2hours. After preincubation with the test compound, HIV-1 (strain NL 4-3)was added to the wells (0.0002 TCID₅₀/cell). After 6 days of additionalincubation, CPE is monitored by MTT conversion. Cytotoxicity is measuredby incubating the drugs for 6 days in the absence of viral inoculation.For transformation of MTT absorbance values into % survival, theabsorbance of uninfected, untreated cells is set to 100% and theabsorbance of infected, untreated cells is set to 0%.

Replication Assay (RA)

The ability of HIV to replicate is monitored in immortalized humanembryonic kidney (293A) cells. These cells are cotransfected with twoplasmids. One plasmid contains a recombinant wild type HIV-1 genome (NL4-3) having its env gene disrupted by a luciferase expression cassette(identified as strain CNDO), the other plasmid contains the env genefrom murine leukemia virus (MLV). These two plasmids provide all theprotein factors in trans to produce a mature chimeric virus having allthe components from HIV-1 except the protein products provided in transfrom the MLV env gene. Virions produced from these cells are infectiousand replicative but cannot produce another generation of infectiousvirions because they will lack the env gene products.

24 hours after transfection, these cells are trypsinized and plated in96 well plates. After the cells have adhered, the media is washed andreplaced with media containing the test compound. Virus production isallowed to proceed for an additional 24 hours. The supernatant is thenharvested and used to reinfect naive 293A cells. Naive cells that areinfected are identified by the luciferase gene product. The number ofluciferase positive cells is a measure of the extent of replicationand/or infection by the recombinant HIV-1. This assay is also adapted totest the resistance to many clinically accepted anti-HIV-1 drugs byusing a HIV-1 genome with several point mutations known to induceresistance to several different classes of anti-HIV drugs. Percentageinhibition is set to 100% for no detectable luciferase positive cellsand 0% for the number of positive cells in infected, untreated controls.

To test the size dependence and sequence independence of PS-ONs onanti-HIV-1 activity, we tested PS-ON randomers that vary in size. CPEassay was conducted in MT4 cells using HIV-1 (strain NL4-3). Infectedcells were treated with increasing concentrations of REP 2004 or REP2006. IC₅₀ values were calculated from linear regressions. Cytotoxicityprofiles in uninfected MT4 cells were determined for REP 2004 and REP2006. We found that as the size of the PS-ON increases, the potency alsoincreases (IC₅₀ decreases). We also noted that the PS-ON randomersexhibited no significant toxicity to the host cells in this assay.

To the best of our knowledge, this is the first time an IC₅₀ for HIV-1as low as 0.011 μM has been reported for a PS-ON.

To more clearly elucidate the effective size range for PS-ON anti-HIV-1activity, we tested more PS-ON randomers covering a broader range ofsizes from 10 to 80 bases by RA using wild-type HIV-I (recombinant NL4-3 (CNDO)). Replication assay was conducted in 293A cells usingrecombinant wild type HIV-1NL4-3 (strain CNDO). In addition, we testedfour protease inhibitors currently used in the clinic (aprenavir,indinavir, lopinavir and saquinavir). Infected cells were treated withincreasing concentrations of Amprenavir, Indinavir, Lopinavir,Saquinavir, REP 2003, REP 2004, REP 2006, and REP 2007. We discoveredthat PS-ON randomers 10 bases and larger have anti-HIV-1 activity andthat the efficacy against HIV-1 also increases with increased PS-ONrandomer length but is saturated at about 40 bases. Moreover, the 40 and80 base PS-ON randomers were almost equivalent in efficacy with the 4clinical controls.

To the best of our knowledge, this is the first time an IC₅₀ for HIV-1as low as 0.014 μM has been reported for a PS-ON.

To test the ability of PS-ON randomers to inhibit a drug resistantstrain of HIV, we duplicated the above test using the recombinant MDRC4strain of HIV-1. This recombinant strain exhibits significant resistanceto at least 16 different clinically accepted drugs from all classes:nucleotide RT inhibitors, non-nucleotide RT inhibitors and proteaseinhibitors. We found that all the PS-ON randomers tested perform as wellagainst the resistant strain as they do against the wild type strain.However, three of the four protease inhibitors show a reduction in theirefficacy against the mutant strain, such that the 40 and 80 base PS-ONrandomers were more potent against this resistant strain than thesedrugs.

Example 5 Inhibition of RSV

The ability of PS-ON randomers to inhibit RSV is measured by monitoringCPE with alamar blue (an indirect measure of cellular metabolism). Humanlarynx carcinoma (Hep2) cells are cultured at 37° C. and 5% CO₂ in MEMplus 5% fetal calf serum. Cells are seeded in 96 well plates at adensity which yields a confluent monolayer of cells after 5-6 days ofgrowth. The day after plating, cells were infected with RSV (strain A2,10^(8.2)TCID₅₀/ml) in the presence of the test compound in a reducedvolume for 2 hours. Following inoculation, the media was changed and wassupplemented with test compound. 6 days after infection, CPE wasmonitored by measuring the fluorescent conversion of alamar blue.Toxicity of test compounds in Hep2 cells was monitored by treatinguninfected cells for 7 days and measuring alamar blue conversion inthese cells. The alamar blue readings in uninfected, untreated cellswere set to 100% survival and the readings in infected, untreated cellswere set to 0% survival.

To confirm the size dependence and sequence independence of PS-ONs onanti-RSV activity, we tested PS-ON randomers that vary in size. Inaddition, we tested the clinically accepted treatment for RSV infection,Ribavirin (Virazole™). CPE assay was conducted in Hep2 cells using RSV(strain A2). Infected cells are treated with increasing concentrationsof REP 2004, REP 2006, REP 2007, or Ribavirin. IC₅₀ values werecalculated from linear regressions are reported in each graph.Cytotoxicity profiles in uninfected Hep2 cells were determined for REP2004, REP 2006, REP 2007, or Ribavirin. We found that as the size of thePS-ON randomer increases, the potency also increases but saturates atabout 40 bases in size. We also noted that 20, 40 and 80 base PS-ONrandomers had IC₅₀ values significantly lower than a clinically acceptedanti-RSV drug, Ribavirin. PS-ON randomers exhibited no toxicity in Hep2cells while Ribavirin was significantly toxic (therapeutic index=2.08).

To the best of our knowledge, this is the first time an IC₅₀ for RSV-1as low as 0.015 μM has been reported for a PS-ON.

Example 6 Inhibition of Coxsackie Virus B2

The ability of PS-ON randomers to inhibit COX B2 is measured monitoringCPE with alamar blue (an indirect measure of cellular metabolism).Rhesus monkey kidney (LLC-MK2) cells are cultured at 37° C. and 5% CO₂in MEM plus 5% fetal calf serum. Cells are seeded in 96 well plates at adensity which yields a confluent monolayer of cells after 5-6 days ofgrowth. The day after plating, cells were infected with COX B2 (strainOhio-1, 10^(7.8)TCID₅₀ml) in the presence of the test compound in areduced volume for 2 hours. Following inoculation, the media was changedand was supplemented with test compound. 6 days after infection, CPE wasmonitored by measuring the fluorescent conversion of alamar blue.Toxicity of test compounds in LLC-MK2 cells was monitored by treatinguninfected cells for 7 days and measuring alamar blue conversion inthese cells. The alamar blue readings in uninfected, untreated cellswere set to 100% survival and the readings in infected, untreated cellswere set to 0% survival.

We tested the anti-COX B2 activity of REP 2006 in the COX B2 CPE assay.The CPE assay was conducted in LLC-MK2 cells using Coxsackievirus B2(strain Ohio-1). Infected cells were treated with increasingconcentrations of REP 2006. The cytotoxicity profile for REP 2006 inLLC-MK2 cells was determined. We found that, while exhibiting someslight toxicity in LLC-MK2 cells, this PS-ON randomer was able topartially rescue infected LLC-MK2 cells from COX B2 infection.

Example 7 Inhibition of Vaccinia Virus

We used the vaccinia infection model as an indicator of the efficacy ofour compounds against poxviruses, including smallpox virus. The abilityof PS-ON randomers to inhibit Vaccinia is measured by monitoring CPEwith alamar blue (an indirect measure of cellular metabolism). Verocells are cultured at 37° C. and 5% CO₂ in MEM plus 5% fetal calf serum.Cells are seeded in 96 well plates at a density which yields a confluentmonolayer of cells after 5-6 days of growth. The day after plating,cells were infected with Vaccinia (10^(7.9)TCID₅₀/ml) in the presence ofthe test compound in a reduced volume for 2 hours. Followinginoculation, the media was changed and was supplemented with testcompound (all at 10 μM, except for Cidofovir which was used at 50 μM).Five days after infection, the supernatants were harvested. The viralload in the supernatant was determined by reinfection of VERO cells withsupernatant diluted 1:100 and the monitoring of CPE 7 days afterreinfection by measuring the fluorescent conversion of alamar blue.

We tested PS-ON randomers that vary in size (REP 2004, 2006 and 2007).In addition, we tested a known effective treatment for Vacciniainfection, Cidofovir (Vistide™). Indirect determination of viral load ininfected supernatants from vaccinia infected VERO cells was determinedby measuring the CPE induced by these supernatants in naive cells. REP2004, 2006 and 2007 were tested at 10 μM while Cidofovir was tested at50 μM. When tested in the Vacinnia CPE assay, we found that treatmentwith REP 2004, 2006 and 2007 all displayed antiviral activity (ie.resulted in supernatants which showed a decreased CPE upon reinfection)but that this activity was weaker than that seen for Cidofovir.

Example 8 Inhibition of DHBV, Parainfluenza-3 Virus, and Hanta Virus

Because DHBV, Parainfluenza-3 virus and Hanta virus do not readilygenerate measurable plaques or CPE, we tested the efficacy of REP 2006in these viruses using a fluorescence focus forming unit (FFFU)detection. In this assay, REP 2006 (at a final concentration of 10 μM)is mixed with the virus which is then adsorbed onto the cells. Afteradsorption, infected cells are allowed to incubate for an additional7-14 days at which point they are fixed in methanol. Regions of viralreplication are detected by immunofluorescence microscopy against theappropriate viral antigen. For each of the three viruses tested, thespecific experimental conditions and results are described in Table 1below: TABLE 1 Inhibition of DHBV, Parainfluenza-3 virus, and Hantavirus. FFFU count Antibody for FFFU FFFU count (10 μM REP Virus CellularHost detection (no drug) 2006) DHBV (HBV Primary duck Mouse anti-DHBV 163 +/− 38.5 0 surrogate) hepatocytes IgG Parainfluenza-3 LLC-MK2 cellsMouse anti-PI3 IgG 288 +/− 126 0 Hanta Virus VERO E6 cells Mouse anti-232.3 +/− 38.17 0 (Strain SinNombre Prospect Hill) nucleoprotein IgG

This initial data shows that at 10 μM, REP 2006 is effective ininhibiting DHBV, Parainfluenza-2 and Hanta Virus. We anticipate thatgiven the robust response in the preliminary test that IC₅₀ values willbe lower. These data support the efficacy of PS-ON randomers for thetreatment of human infections of Hanta Virus and Hepatitis B (closelyrelated to DHBV) as well as providing a rationale for the immediatetreatment of pediatric bronchiolitis caused by RSV and Parainfluenza-3,which may not require differential diagnosis for treatment to begin.

Example 9 Currently Non-Responsive Viruses

To date we have not observed a detectable anti-viral efficacy with PS-ONrandomers (up to 10 μM) without using a delivery system, a drugcombination, or a chemical modification in the following viral systemsdescribed in Table 2: TABLE 2 Viral Systems Assay Virus Strain CellularHost paradigm Corona virus MHV2 (mouse) NCTC-1496 cells Plaque (SARSsurrogate) MHV-A59 (mouse) DBT cells reduction HCoV-OC43 HRT-18 cells(human) BVDV (HCV NA BT cells CPE by surrogate) alamar blue RhinovirusHGP HeLa cells CPE by alamar blue Adenovirus Human Ad5 293A cells Plaquereduction

Under the current testing procedures, we did not demonstrate activity.However, the lack of demonstrated antiviral activity may be due tolimitations of the particular assays used. Additional testing isunderway to demonstrate efficacious results with these viruses.

Since our evidence indicates that the charge characteristics of a PS-ONare important for the inhibition of viruses from several differentfamilies, we expect that this charge dependent mechanism for theinhibition of viral activity has the potential to inhibit the activityof all encapsidating viruses. The corollary to this is that the lack ofdetected anti-viral efficacy against those viruses listed in Example 9suggests that the interaction between the PS-ON and the structuralproteins of these viruses may not strong be enough to prevent theinteraction of viral proteins during the replication of these viruses.In this case, one way of achieving efficacy against these viruses is toalter the charge characteristics of the DNA or anti-viral polymer (e.g.,substituting phosphorodithioate for phosphorothioate linkages in DNA) sotheir affinity for viral proteins is increased.

Example 10 Inhibition of Influenza A

The ability of PS-ONs to inhibit the influenza virus (INF) A is measuredin a plaque reduction assay (PRA). Immortalized Canine kidney (MDCK)cells are cultured at 37° C. and 5 CO₂ in MEM plus 10% fetal calf serumsupplemented with gentamycin, vancomycin and amphoterecin B. Cells areseeded in 6 well plates at a density which yields a confluent monolayerof cells after 6 days of growth. Upon reaching confluency, the media ischanged to contain only supplements as described above and cells arethen exposed to INF A (strain H3N2, approximately 35-70 PFU total) inthe presence of the test compound for 60 minutes. After viral exposure,the media is replaced with new media containing drug only. 24 hoursafter infection, media is again replaced with overlay media containing4% albumin, 0.025% DEAE dextran, 2 mg/ml TPCK-treated trypsin and 0.8%seaplaque agarose, supplements as described above and no test compound.Plaque counting is performed 2-3 days post infection following formalinfixation and cresyl violet staining of infected cultures.

We tested the anti INF A activity of a variety of PS-ON randomers in theINF A PRA assay. We found that only REP 2006 showed any measurableantiviral activity but that this activity was significant (see followingtable 3). TABLE 3 Activity of PS-ON randomers against INF A (H3N2).Randomer IC₅₀ (μM) REP 2003 >10 REP 2004 >10 REP 2006 ˜3

Since only the largest randomer seemed to have any activity and we knowthat the activity of randomers in many other viruses was size dependent,we tested the antiviral activity of a larger size distribution ofrandomers using a broader dilution range. We discovered that as forother viruses we had tested, the anti-INF A activity of randomers becamemore potent as their length increased but that no significant increasein activity was seen for randomers above 40 bases in length. TABLE 4Size dependent anti-INF A activity of PS-ON randomers. Randomer IC₅₀(μM) REP 2032 >50 REP 2003 >50 REP 2004 ˜25 REP 2005 ˜6.25 REP 2006˜1.25 REP 2007 ˜0.625

To determine the mechanism of action of REP 2006 we attempted todetermine the effect of adding REP 2006 (at IC99 concentration) atvarious times before, during and after infection. In this experiment, weobserved that even 5 hours (300 min) after infection, adding REP 2006resulted in a complete inhibition of INF A activity (see followingtable). These results indicate that at least a significant portion ofthe action of REP 2006 against influenza occurs post infection. SincePS-ON randomers do not readily enter the cell, PS-ON randomers may alsointerfere with viral budding from the host cell. TABLE 5 Time ofaddition of REP 2006 versus effect on INF A activity. Time of REP 2006mixing with virus relative to Infectivity infection (min) (%) no drug(ctl) 100 −30 0  −5 0  0 0  5 0  30 0  60 0  90 0 120 0 180 0 240 0 3000

Example 11 Tests for Determining if an Oligonucleotide ActsPredominantly by a Sequence Independent Mode of Action

We have shown herein that the antiviral activity of the present ONsoccurs by a sequence-independent mode of action. Of course a personskilled in the art could prepare sequence-specific ONs, for example anantisense ON targeting a mRNA of a particular virus and incorporatingall phosphorothioate and 2′ O-methyl modifications. However such an ONwould have benefited from the ON modifications we have described hereinand the fact that we have demonstrated herein that the activity of sucha modified ON is sequence independent. Thus, an ON shall be consideredto have sequence-independent activity if it meets the criteria of anyone of the 5 tests outlined below, i.e., if a substantial part of itsfunction is due to a sequence-independent activity. The ONs used in thefollowing tests can be prepared following the general methodologydescribed in example 12 for the synthesis of PS-ONs.

Test #1—Effect of Partial Degeneracy of a Candidate ON on its AntiviralEfficacy

This test serves to measure the antiviral activity of a candidate ONsequence when part of its sequence is made degenerate. If the degenerateversion of the candidate ON having the same chemistry retains itsactivity as described below, is it deemed to have sequence-independentactivity. Candidate ONs will be made degenerate according to thefollowing rule:

-   -   L=the number of bases in the candidate ON    -   X=the number of bases on each end of the oligo to be made        degenerate (but having the same chemistry as the candidate ON)    -   If L is even, then X=integer (L/4)    -   If L is odd, then X=integer ((L+1)/4)    -   X must be equal to or greater than 4

If the candidate ON is claimed to have an anti-viral activity against amember of the herpesviridae, retroviridae, or paramyxoviridae families,the IC₅₀ generation will be performed using the assay described hereinfor that viral family preferably using the viral strains indicated. Ifthe candidate ON is claimed to have an anti-viral activity against amember of a particular virus family not mentioned above, then the IC₅₀values shall be generated by a test of antiviral efficacy accepted bythe pharmaceutical industry. IC₅₀ values shall be generated using aminimum of seven concentrations of compound, with three or more pointsin the linear range of the dose response curve. Using these tests, theIC₅₀ of the candidate ON shall be compared to its degeneratecounterpart. If the IC₅₀ of the partially degenerate ON is less than5-fold greater than the original candidate ON (based on minimumtriplicate measurements, standard deviation not to exceed 15% of mean)then the ON shall be deemed to act predominantly by a sequenceindependent mode of action.

Test #2—Comparison of Antiviral Activity of a Candidate ON with an ONRandomer.

This test serves to compare the anti-viral efficacy of a candidate ONwith the antiviral efficacy of a randomer ON of equivalent size andchemistry in the same virus.

If the candidate ON is claimed to have an anti-viral activity against amember of the herpesviridae, retroviridae, or paramyxoviridae families,the IC₅₀ generation will be performed using the assay described hereinfor that viral family preferably using the viral strains indicated. Ifthe candidate ON is claimed to have an anti-viral activity against amember of a particular virus family not mentioned above, then the IC₅₀values shall be generated by a test of antiviral efficacy accepted bythe pharmaceutical industry. IC₅₀ values shall be generated using aminimum of seven concentrations of compound, with three or more pointsin the linear range of the dose response curve. Using this test, theIC₅₀ of the candidate ON shall be compared to an ON randomer ofequivalent size and chemistry. If the IC₅₀ of the ON randomer is lessthan 5-fold greater than the candidate ON (based on minimum triplicatemeasurements, standard deviation not to exceed 15% of mean) then thecandidate ON shall be deemed to act predominantly by a sequenceindependent mode of action.

Test #3—Comparison of Antiviral Activity of a Candidate ON in TwoNon-Homologous Viruses from the Same Viral Family

This test serves to compare the efficacy of a candidate ON against atarget virus whose genome is homologous to the candidate ON with theefficacy of the candidate ON against a second virus whose genome has nohomology to that candidate ON but is in the same viral family. Forexample, if a candidate ON is reported to have activity against HSV, itsactivity against HSV will be compared to its activity against CMV or VZVetc. The comparison of the relative activities of the candidate ON inthe target virus and the second virus is accomplished by using theactivities of an ON randomer of the same length and chemistry in bothviruses to normalize the IC₅₀ values for the candidate ON obtained inthe two viruses.

Thus, if the candidate ON is claimed to have an anti-viral activityagainst a certain virus, then the IC₅₀ generation will be determined inthis virus using one of the assays described herein for theherpesviridae, retroviridae, or paramyxoviridae families, or otherassays known in the art. Similarly, IC₅₀ generation will be performedfor the candidate ON against a second virus using one of the assays asdescribed herein or an assay accepted by the industry for a virus whosegenome has no homology to the sequence of the candidate ON but is fromthe same viral family. IC₅₀ generation is also performed for a randomerof equivalent size and chemistry against each of the viruses. The IC₅₀of the ON randomer against the two viruses are used to normalize theIC₅₀ values for the candidate ON against the two viruses as follows:

-   -   An equivalent algebraic transformation is applied to the IC₅₀ of        the candidate ON and the ON randomer in the first (homologous)        virus such that the IC₅₀ of the randomer is now 1.    -   An equivalent algebraic transformation is applied to the IC₅₀ of        the candidate ON and the ON randomer in the second        (non-homologous) virus such that the IC₅₀ of the randomer is now        1.    -   The fold difference in the IC₅₀s for the candidate ON in the        homologus versus the non-homologous virus is calculated by        dividing the transformed IC₅₀ of the candidate ON in the        non-homologous virus by the transformed IC₅₀ of the candidate ON        in the homologous virus.

The candidate ON shall be deemed to act predominantly by a sequenceindependent mode of action if the fold difference in IC₅₀ between thetwo viruses is less than 5.

Test #4: Antiviral Activity of a Candidate ON in a Different ViralFamily

This test serves to determine if a candidate ON has a drug-like activityin a virus where the sequence of the candidate ON is not homologous toany portion of the viral genome and the virus is from a differentfamily. Thus the candidate ON shall be tested using one of the assaysdescribed herein for the herpesviridae, retroviridae or paramyxoviridaesuch that the sequence of the candidate ON tested is not homologous toany portion of the genome of the virus to be used. An IC₅₀ value shallbe generated using a minimum of seven concentrations of the candidateON, with three or more points in the linear range. If the resulting doseresponse curve indicates a drug-like activity (which can typically beseen as a decay or sigmoidal curve, having reduced anti-viral efficacywith decreasing concentrations of candidate ON) and the IC₅₀ generatedfrom the curve is less than 10 μM, the candidate ON shall be deemed tohave a drug-like activity. If the candidate ON is deemed to have adrug-like activity in a virus from a different family for which thecandidate ON is not complementary and thus can have no sequencedependent antisense activity, it shall be considered to actpredominantly by a sequence independent mode of action.

Test #5. Extracellular Antiviral Activity of a Candidate ON

Our current results indicate that the sequence-independent antiviralactivity of ONs occurs outside the cell. The state of the art in ONtechnology teaches that, since ONs are not readily cell permeable, theymust be delivered across the cell membrane by an appropriate carrier tohave antisense activity. Thus, the antiviral activity of antisense ONsby definition is dependent on delivery inside cells for activity. If aparticular sequence-specific candidate ON has in vitro antiviralactivity when used naked (and therefore having poor intracellularpenetration), it must benefit from the sequence-independent propertiesof ONs described in this invention.

If the sequence-specific candidate ON is complementary to a portion ofthe genome of HSV-1, HIV-1 or RSV, then the presence of asequence-independent antiviral activity of the candidate ON shall bedetermined in the appropriate assay described below. If the candidate ONis complementary to a virus which is not HSV-1, HIV-1 or RSV, then theantiviral activity of the candidate ON shall be determined using anassay accepted by the pharmaceutical industry.

Using the appropriate assay, the antiviral activity of the nakedcandidate ON shall be compared to that of the encapsulated (fortransfection) candidate ON (using identical candidate ON concentrationsin both naked and encapsulated conditions). The activity shall bemeasured by a dose response curve with not less than 7 concentrations,at least 3 of which fall in the linear range which includes the 50%inhibition of viral activity. The IC₅₀ (the concentration which reducesviral activity by 50%) shall be calculated by linear regression of thelinear range of the dose response curve as defined above. If the IC₅₀ ofthe naked candidate ON is less than 5-fold greater than that of theencapsulated candidate ON, then the activity of the candidate ON shallbe deemed to act predominantly by a sequence independent mode of action.

Thresholds Used in These Tests

The purpose of these tests are to determine by a reasonable analysis, ifONs benefit from or utilize the sequence-independent antiviralproperties of ONs which we have described herein and is acting withsequence-independent activity. Of course anyone skilled in the art willrealize that, given the inherent variability of all testingmethodologies, especially antiviral testing methods, a determination ofdifferences in antiviral activity between two compounds may not bereliably concluded if the threshold is set at a 2 or 3 fold differencebetween the activities of said compounds. This is due to the fact thatvariations from experiment to experiment with the same compound in theseassays can yield IC₅₀s which vary in this range. Thus, to be reasonablycertain that real differences between the activities of two compounds(e.g. two ONs) exist, we set a threshold of at least a 5-fold differencebetween the IC₅₀s of said compounds. This threshold ensures thereliability of the assessment of the above mentioned tests.

The thresholds described in tests 1 to 3 and 5 above are the defaultthresholds. If specifically indicated, other thresholds can be used inthe comparison tests 1 to 3 and 5 described above. Thus for example, ifspecifically indicated, the threshold for determining whether an ON isacting with sequence-independent activity can be any of 10-fold, 8-fold,6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1.5-fold, or equal. Thethreshold described in test 4 above is also a default threshold. Ifspecifically indicated, the threshold for determining whether an ON hassequence-independent activity in test 4 can be an IC₅₀ of less than 10μM, 5 μM, 1 μM, 0.8 μM, 0.6 μM, 0.5 μM, 0.4 μM, 0.3 μM, 0.2 μM or 0.1μM.

Similarly, though the default is that satisfying any one of the above 5tests is sufficient, if specifically indicated, the ON can be requiredto satisfy any two (e.g., tests 1 & 2, 1 & 3, 1 & 4, 1 & 5, 2 & 3, 2& 4,2 & 5, 3 & 4, and 3 & 5), any three (e.g., tests 1 & 2 & 3,1 & 2 & 4,1,& 2 & 5, 1 & 3 & 4, 1 & 3 & 5, 2 & 3 & 4, and 2 & 4 & 5), any 4 of thetests (e.g., 1 & 2 & 3 & 4, 1 & 2 & 3 & & 5, and 2 & 3 & 4 & 5) at adefault threshold, or if specifically indicated, at another threshold(s)as indicated above.

Example 12 Methodologies

The following methods are provided for application in the testsdescribed in example 11.

Oligonucleotide Synthesis

The present oligonucleotides can by synthesized using methods known inthe art. For example, unsubstituted and substituted phosphodiester (P═O)oligonucleotides can be synthesized on an automated DNA synthesizer(e.g., Applied Biosystems model 380B or Akta Oligopilot 100) usingstandard phosphoramidite chemistry with oxidation by iodine.Phosphorothioates (P═S) can be synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle can be replaced by0.2 M solution of 311-1,2-benzodithiole-3-one 1,1-dioxide inacetonitrile for the step-wise thioation of the phosphite linkages. Thethioation wait step can be increased to 68 sec, followed by the cappingstep. After cleavage from the support column and deblocking inconcentrated ammonium hydroxide at 55° C. (18 h), the oligonucleotidescan be purified by precipitating twice with 2.5 volumes of ethanol froma 0.5 M NaCl solution.

Antiviral Assay for Herpesviridae

A plaque reduction assay for herpesviridae is performed as follows:

For HSV-1 or HSV-2, VERO cells (ATCC# CCL-81) are grown to confluence in12 well tissue culture plates (NUNC or equivalent) at 37° C. and 5% CO₂in the presence of MEM supplemented with 10% heat inactivated fetal calfserum and gentamycin, vancomycin and amphoterecin B. Upon reachingconfluency, the media is changed to contain 5% fetal calf serum andantibiotics as detailed above supplemented with either HSV-1 (strainKOS, 40-60 PFU total) or HSV-2 (strain MS2, 40-60 PFU total). Viraladsorbtion proceeds for 90 minutes, after which cells are washed andreplaced with new “overlay” media containing 5% fetal calf serum and 1%human immunoglobins. Three to four days after adsorbtion, cells arefixed by formalin and plaques are counted following formalin fixationand cresyl violet staining.

For CMV, human fibroblasts are grown as specified for VERO cells in theHSV-1/2 assay. Media components and adsorbtion/overlay procedures areidentical with the following exceptions:

-   -   1. CMV (strain AD169, 40-60 PFU total) is used to infect cells        during the adsorbtion.    -   2. In the overlay media, 1% human immunoglobins are replaced by        4% sea-plaque agarose.

For other herpesviridae, testing is to be conducted in the plaque assaydescribed above using an appropriate cellular host and 40-60 PFU ofvirus during the adsorbtion.

This test is only valid if identifiable plaques are present in theabsence of compound at the end of the test.

In this test, IC₅₀ is the concentration at which 50% of the plaques arepresent compared to the untreated control.

Compound to be tested is present during the adsorption and in theoverlay.

Antiviral Assay for Retroviridae

Assaying for the retroviridae HIV-1 is performed by detection of totalp24 in the supernatant of HIV-1 infected cells by ELISA is performed asfollows:

PBMCs were isolated from fresh human blood obtained from screeneddonors, seronegative for HIV and HBV. Peripheral blood cells werepelleted/washed 2-3 times by low speed centrifugation and resuspensionin PBS to remove contaminating platelets. The washed blood cells werethen diluted 1:1 with Dulbecco's phosphate buffered saline (PBS) andlayered over 14 mL of Lymphocyte Separation Medium (LSM; celigro® byMediatech, Inc.; density 1.078±0.002 g/ml; Cat.# 85-072-CL) in a 50 mLcentrifuge tube and centrifuged for 30 minutes at 600×g. Banded PBMCswere gently aspirated from the resulting interface and subsequentlywashed 2× with PBS by low speed centrifugation. After the final wash,cells were counted by trypan blue exclusion and resuspended at 1×10⁷cells/mL in RPMI 1640 supplemented with 15% Fetal Bovine Serum (FBS), 2mM L-glutamine, 4 μg/mL PHA-P. The cells were allowed to incubate for48-72 hours at 37° C. After incubation, PBMCs were centrifuged andresuspended in RPMI 1640 with 15% FBS, 2 mM L-glutamine, 100 U/mLpenicillin, 100 μg/mL streptomycin, 10 μg/mL gentamycin, and 20 U/mLrecombinant human IL-2. PBMCs were maintained in this medium at aconcentration of 1-2×10⁶ cells/mL with biweekly medium changes untilused in the assay protocol. Monocytes were depleted from the culture asthe result of adherence to the tissue culture flask.

For the standard PBMC assay, PHA-P stimulated cells from at least twonormal donors were pooled, diluted in fresh medium to a finalconcentration of 1×10⁶ cells/mL, and plated in the interior wells of a96 well round bottom microplate at 50 μL/well (5×10⁴ cells/well). Testdrug dilutions were prepared at a 2× concentration in microtiter tubesand 100 μL of each concentration was placed in appropriate wells in astandard format. After a 2-hr preincubation period (cells+drug), 50 μLof a predetermined dilution of virus stock was placed in each test well(final MOI 0.1). Wells with cells and virus alone were used for viruscontrol. Separate plates were prepared identically without virus fordrug cytotoxicity studies using an MTS assay system (described below).The PBMC cultures were maintained for seven days following infection, atwhich time cell-free supernate samples were collected and assayed forreverse transcriptase activity as described below.

P24 ELISA kits were purchased from Coulter Electronics. The assay isperformed according to the manufacturer's instructions. Control curvesare generated in each assay to accurately quantify the amount of p24antigen in each sample. Data are obtained by spectrophotometric analysisat 450 nm using a Molecular Devices Vmax plate reader. Finalconcentrations are calculated from the optical density values

This test is only valid if there is an accumulation of p24 in the tissueculture supernatant in the infected, untreated cells.

In this test, IC₅₀ is the concentration at which the amount of p24detectable is 50% of the p24 present in the untreated control.

Compound to be tested is present during the adsorption and in the mediaafter adsorption.

Antiviral Assay for Paramyxoviridae

For RSV, a measurement of CPE is performed as follows:

Hep2 cells were plated in 96 well plates and allowed to grow overnightin MEM plus 5% fetal calf serum at 37° C. and 5% CO₂. The next day,cells are infected with RSV (strain A2, 10^(8.2) TCID50/ml in 100ul/well) by adsorbtion for 2 hours. Following adsorbtion, media ischanged and after 7 days growth, CPE is measured by conversion of AlamarBlue dye to its fluorescent adduct by living cells.

This test is only valid if CPE measurement (as measured by Alamar Blueconversion) in infected cells in the absence of compound is 10% of theconversion measured in uninfected cells.

For purposes of IC₅₀ comparison, 100% CPE is set at the conversion levelseen in infected cells and 0% CPE is set at the conversion seen inuninfected cells. Therefore IC₅₀ is the concentration of compound whichgenerates 50% CPE.

Compound to be tested is present during the adsorption and in the mediaafter adsorption.

Example 13 2′-O Methylated Phosphorothioated Randomers Exhibit PotentAntiviral Activity with Increased pH Resistance and Lower Serum ProteinBinding

We show herein that PS-ON randomers do not act via a sequence specificmechanism (i.e. their activity does not require them to bind to nucleicacid and their activity is not due to a sequence specific aptamericeffect). We further show in this example the effect of oligonucleotidescombining unmodified linkages, phosphorothiate linkages, 2′-O methylmodified riboses and unmodified ribonucleotides on a 40 base randomerwith respect to their antiviral activity, serum protein interaction andchemical stability.

All randomers were prepared using standard solid phase, batch synthesisat the University of Calgary Core DNA Services lab on a 1 or 15 molsynthesis scale, deprotected and desalted on a 50 cm Sephadex G-25column.

For antiviral activity testing in influenza A (INF A), immortalizedCanine kidney (MDCK) cells are cultured at 37° C. and 5% CO₂ in MEM plus10% fetal calf serum supplemented with gentamycin, vancomycin andamphoterecin B. Cells are seeded in 6 well plates at a density whichyields a confluent monolayer of cells after 6 days of growth. Uponreaching confluency, the media is changed to contain only supplements asdescribed above and cells are then exposed to INF A (strain H3N2,approximately 35-70 PFU total) for 60 minutes. After viral exposure, themedia is replaced with new media containing drug only. Plaque countingis performed 2-3 days post infection following formalin fixation andcresyl violet staining of infected cultures.

For antiviral testing in HSV, immortalized African Green Monkey kidney(VERO) cells are cultured at 37° C. and 5% CO₂ in MEM plus 10% fetalcalf serum supplemented with gentamycin, vancomycin and amphoterecin B.Cells are seeded in 12 well plates at a density which yields a confluentmonolayer of cells after 4 days of growth. Upon reaching confluency, themedia is changed to contain only 5% serum plus supplements as describedabove and cells are then exposed to HSV-1 (strain KOS, approximately40-60 PFU total) in the presence of the test compound for 90 minutes.After viral exposure, the media is replaced with new “overlay” mediacontaining 5% serum, 1% human immunoglobulins, supplements as describedabove and the test compound. Plaque counting is performed 3-4 days postinfection following formalin fixation and cresyl violet staining ofinfected cultures.

To determine serum protein interaction, a phosphorothioate randomerlabeled at the 3′ end with FITC (the bait) is diluted to 2 nM in assaybuffer (10 mM Tris, pH7.2, 80 mM NaCl, 10 mM EDTA, 100 mMb-mercaptoethanol and 1% tween 20). This oligo is then mixed with theappropriate amount of non heat-inactivated FBS. Following randomer-FBSinteraction, the complexes are challenged with various unlabelledrandomers to assess their ability to displace the bait from its complex.Displaced bait is measured by fluorescence polarization. Thedisplacement curve was used to determine Kd.

pH resistance was determined by incubation of randomers in PBS adjustedto the appropriate pH with HCl. 24 hours after incubation, samples wereneutralized with 1M TRIS, pH 7.4 and run on denaturing acryalmide gelsand visualized following EtBr staining.

For these experiments, we compared the behaviours of different modifiedrandomers: REP 2006, REP 2024, REP 2107, REP 2086 and REP 2060 (seeTable 6 in this example). The antiviral activities of these randomerswere tested for antiviral activity in HSV and influenza A by plaquereduction assay (see Table 7 in this example). In these two viruses, REP2006, 2024 and 2107 had similar and potent anti-viral activity, REP 2060showed significant anti-HSV activity and REP 2086 had no detectableantiviral activity in either HSV-1 or influenza A under these assayconditions. TABLE 6 Randomer description Randomer Description (N = A, G,T/U or C) REP 2006 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN REP 2024NNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNN REP 2107NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN REP 2086NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN REP 2060NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN = unmodified deoxyribonucleotide, unmodified linkageN = unmodified deoxyribonucleotide, phosphorothiate linkageN = 2′-O methyl modified ribose, unmodified linkageN = 2′-O methyl modified ribose + phosphorothioate linkageN = unmodified ribonucleotide + phosphorothioate linkage

TABLE 7 Antiviral activity of various randomers in HSV and influenza ARandomer IC₅₀ (μM) (HSV) IC₅₀ (μM) (influenza A) REP 2006 0.1 ˜2 REP2024 0.14 ˜1.5 REP 2107 0.085 ˜1 REP 2086 No activity no activity REP2060 0.85 Not tested

The relative affinity of these randomers for serum proteins wasdetermined as described above. The results of these experiments showedthat REP 2107 has a lower affinity to serum proteins than REP 2006 orREP 2024 (see Table 8 in this example) and that there was no interactiondetected between REP 2086 and serum proteins. Moreover, at saturation ofcompetition, REP 2107 was less effective at displacing bound bait thanREP 2006 or REP 2024 (see Table 9 in this example). TABLE 8 Serumprotein affinity of various randomers. Randomer Kd (nM) (FBS) 2006 132024 13 2107 27 2086 no binding

TABLE 9 Displacement of bait randomer at saturation. Randomer %displaced bait 2006 75 2024 80 2107 60 2086 no displacement

Finally, we tested the pH stability of these randomers in the range ofpH 1 to pH 7 over 24 hours of incubation at 37° C. While REP 2006 andREP 2024 showed significant degredation at pH 3 and complete degredationat pH 2.5, REP 2107, 2086 and 2060 were completely stable at pH 1 after24 h of incubation.

These results duplicate our previous findings that thephosphorothioation of ON randomers is highly beneficial for theirantiviral activity. We further demonstrate here that the incorporationof 2′-O methyl modifications in PS-ON randomers does not affect theantiviral activity of these molecules, even when every ribose in thePS-ON randomer contains a 2′-O-methyl modification. Moreover, the fully2′-O-methylated, fully phosphorothioated randomer (REP 2107) has aweaker interaction with serum proteins and shows a significantlyincreased resistance to low pH induced hydrolysis

Example 14 PS-ONs Act by a Predominantly Extracellular Mode of Action

Prior art has taught that the use of delivery agents to increase theintracellular concentrations of PS-ONs would be beneficial to theiractivity. We demonstrate here that the antiviral activity of PS-ONs actspredominantly outside the cell and therefore would not receive a majorbenefit from the transfection enhancement of an intracellular deliveryagent.

In this example, we use a PS-ON made of deoxyribonucleotides (DNA)without other modifications such as ribonucleotides (RNA) or 2′-O-methylmodification. It is safe to consider that this data will apply to PS-ONbearing additional modifications because it is known is the art thatthese molecules do not penetrate cells in vitro easily without the aideof a delivery system or a tranfection agent, especially in cases ofantisense antivity.

For the determination of cellular delivery, HeLa cells were culturedunder standard conditions and then incubated with fluorescently labelledREP 2006 (FL-REP2006, a 3′ fluorescein isothiocynate conjugated 40 basePS-ON randomer), either naked or encapsulated with a delivery agent (inthis case DOTAP [1,2-Dioleoyl-3-Trimethylammonium-Propane], a cationiclipid). After various times of incubation, cells were thoroughly washedwith PSB to remove any non-internalized ON and the cells weresubsequently lysed. The level of intracellular ON in the cell lysate wasdetermined using a fluorescence plate reader.

The determination of antiviral efficacy with naked, DOTAP and PEI(polyethylene imine) encapsulated REP 2006 in HSV-1 and influenza wasdetermined as described above.

The determination of the time of action of REP 2006 during theinfectious cycle of HSV-1 was determined as described above, but addingREP 2006 at various times before, during and after infection. In HIV-1,this was determined by adding REP 2006 to HIV-LTR-beta-gal HeLa cells atvarious time before, during and after infection. HIV-1 infection wasmonitored by a colourmetric assay of beta-gal production usingabsorbance spectroscopy.

We first determined that DOTAP and PEI could deliver fluorescent REP2006 inside cells (see table 10). This data showed that both DOTAP andPEI were capable of delivering FL-2006 (and by inference REP 2006)inside cells. TABLE 10 Intracellular concentration of FL-REP 2006 withand without delivery (pmol/cell) Incubation FL-REP 2006 + timeFL-REP2006 DOTAP FL-REP2006 + PEI  1 hour 6 × 10⁻⁵ 5 × 10⁻⁴ 6 × 10⁻⁵  6hours 6 × 10⁻⁵ 9 × 10⁻⁴ 3 × 10⁻⁴ 24 hours 6 × 10⁻⁵ 1.7 × 10⁻³   7 × 10⁻⁴

We then determined the activity of encapsulated (DOTAP or PEI) REP2006in HSV-1 influenza A (see Table 11 and 12 in this example) These resultsshowed that encapsulated REP 2006 had no detectable antiviral activityin both HSV-1 and influenza. TABLE 11 Activity of encalsulated REP 2006in HSV-1 (IC₅₀, μM) REP2006 REP2006 + DOTAP REP 2006 + PEI 0.074 Noactivity No activity

TABLE 12 Activity of PEI encapsulated REP 2006 in influenza A (%inhibition of plaque formation) ON concentration (μM) REP 2006 REP2006 + PEI 0 0 0 0.625 0 0 0.125 50 0 2.5 75 0 5 100 0 10 100 0

Finally, a time of addition study in HSV-1 and HIV-1 was performed whereREP 2006 was added at various times before, during and after theinfection. These results showed that in both viruses, REP 2006 was mosteffective when present before or during the infection, indicating thatit was a fusion/entry inhibitor in HSV-1 and HIV-1.

These results demonstrate that the antiviral activity or REP 2006 andPS-ONs bearing additional modifications such as, but withoutrestriction, ribonucleotides (RNA) or 2′-O-methyl, occurs principallyoutside the cell.

Example 15 REP 2107 Exhibits Superior Nuclease Resistance

40 mer randomers of various chemistries were assessed for their abilityto resist degredation by various nucleases for 4 hours at 37° C. (seeTable 13 in this example). While most chemistries exhibited resistanceto more than one nuclease, only REP 2107 was resistant to all fournucleases tested. It is important to note that REP 2024 (which has 2′-Omethyl modifications at the 4 riboses at each end of the molecule)showed the same resistance profile as its parent molecule REP 2006,being sensitive to S1 nuclease degradation while 2107 (fully 2′-O methylmodified) was resistant to this enzyme. These results suggest that REP2107 will be the most effective of the tested oligonucleotides inresisting degredation by nucleases in the blood. TABLE 13 Resistance tovarious nucleases by different randomer chemistries. Sensitive (S) orResistant (R) after 4 h incubation at 37° C. Phosphodiesterase S1Nuclease Bal 31 Exonuclease I II (Fermentas (NEB (NEB Randomer (SigmaP9041) #EN0321) M0213S) M0293S) REP2015 R S S S REP2107 R R R R REP2006R S R R REP2086 R R S R REP2024 R S R R

Example 16 Phosphorothioated Polypyrimidine ONs Exhibit Acid andNuclease Resistance

To determine the extent of ONs acid resistance of ONs, various 40 baseONs having different chemistries and/or sequences are incubated in PBSbuffered to different pH values for 24 hours at 37° C. The degradationof these ONs was assessed by urea-polyacryamide gel electrophoresis (seetable 14).

The results of these studies show that randomer ONs (containing bothpyrimidine and purine nucleotides) are resistant to acidic pH only whenthey were fully 2′-O-methylated. Our data indicated that even partially2′-O-methylated ONs (gapmers, REP 2024) do not display any significantincrease in acid resistance compared to fully phosphorothioated ONs.Even fully phosphorothioated randomers show no increased pH resistancecompared to unmodified ONs. In contrast, we noted that thephosphorothioated 40mer ONs containing only the pyrimidine nucleotidescytosine (polyC, REP 2031) or thymidine (polyT, REP 2030) or the polyTCheteropolymer (REP 2056) had equivalent acid resistance compared to thefully 2′-O-methylated randomers whether phosphorothioated (REP 2107) ornot (REP 2086). Contrary to the results for the polypyrimidineoligonucleotides, phosphorothioated oligonucleotides containing only thepurine nucleotide adenosine (polyA, REP 2029) or any adenosine orguanosine nucleotides (REP 2033, 2055, 2057) showed no greater acidresistance compared to unmodified DNA.

These results are significant because the preferred way described in theprior art to achieve greater acid resistance compared tophosphorothioated ONs was to add 2′-O-methyl modifications (or other2′-ribose modifications) or other modifications. The present datademonstrates that the 2′-O-methyl ribose modification or other 2′-ribosemodifications are not required if the ON is a polypyrimidine (i.e.contains only pyrimidine nucleotides [e.g. homopolymers of cytosine orthymidine or a heteropolymer of cytosines and thymidines]) to achieve pHand nuclease resistance. The presence of purines (A or G) even in thepresence of pyrimidines, can render ONs acid labile. TABLE 14 Acidstability of various 40 mer ONs stability to various pHs after 24 h at37° C. pH ON name sequence modification pH 1 pH 2 2.5 pH 3 pH 4 pH 5 pH7 REP 2015 randomer none − − −/+ + +++ +++ +++ REP 2006 randomer PS − −−/+ + +++ +++ +++ REP 2086 randomer 2′OMe +++ +++ +++ +++ +++ +++ +++REP 2107 randomer PS, 2′OMe +++ +++ +++ +++ +++ +++ +++ REP 2024randomer PS, 2′OMe − − −/+ + +++ +++ +++ gapmer REP 2031 polyC PS ++++++ +++ +++ +++ +++ +++ REP 2030 polyT PS +++ +++ +++ +++ +++ +++ +++REP 2029 polyA PS − − − − ++ +++ +++ REP 2033 polyTG PS − − − − ++ ++++++ REP 2055 polyAC PS − − − − ++ +++ +++ REP 2056 polyTC PS +++ +++ ++++++ +++ +++ +++ REP 2057 polyAG PS − − − − ++ +++ +++PII = phosphodiesterase II,S1 = S1 nuclease,Exo1 = Exonuclease 1,PS = all linkages phosphorothioated,2′OMe = all riboses are 2′O methylated.+++ = no degradation,++ = less than 5-% degradation,−/+ = more than 90% degradation,− = completely degraded

To determine the extent of ON nucleotide composition and modificationson nuclease resistance, various 40 base ONs having different nucleotidecompositions and modifications were incubated in the presence of variousendo and exonucleases for 4 hours at 37° C. The degradation of these ONswas assessed by urea-polyacryamide gel electrophoresis.

The results of these studies showed that randomer ONs were resistant toall four enzymes tested (phosphodiesterase II [Sigma], S1 nuclease[Fermentas], Bal31 [New England Biolabs] and exonuclease 1 [New EnglandBiolabs]) only when they were fully phosphorothioated and fully 2′-O-methylated (see table 15). Omission of any of these modifications inrandomers resulted in increased sensitivity to one or more of thenucleases tested. We noted that the fully phosphorothioated, partially2′-O -methylated randomer (REP 2024) was equivalent in nucleaseresistance to REP 2006, indicated that 2′-O-methylation may be requiredon each nucleotide of a phosphorothioated ON to achieve the optimalnuclease resistance. However, we noted that the phosphorothioated 40merpolypyrimidine poly cytosine (poly C, REP 2031) had equivalent nucleaseresistance compared to the fully phosphorothioated, fully 2′O methylatedrandomer (REP 2107).

These results are significant because the prior art teaches that thepreferred way to enhance nuclease resistance of phosphorothioated ONs isto add 2′-O-methyl modifications, other 2′-ribose modifications, orother modifications. This new data demonstrates that the 2′-O-methylmodification or other 2′-ribose modifications or any other modificationsare not required to enhance nuclease resistance if the ON is fullyphosphorothioated and consists of a homopolymer of pyrimidines. TABLE 15Nuclease resistance of various 40 mer ONs Nuclease resistance ON after 4h at 37° C. name sequence modification PII S1 Bal 31 Exo 1 REP randomernone − − − − 2015 REP randomer PS +++ − ++++ ++++ 2006 REP randomer2′OMe ++++ ++++ − ++++ 2086 REP randomer PS, 2′OMe ++++ ++++ ++++ ++++2107 REP randomer PS, 2′OMe ++++ − ++++ ++++ 2024 gapmer REP polyC PS++++ ++++ ++++ ++++ 2031 REP2029 Poly A PS − − ++++ ++++ REP2030 Poly TPS − − ++++ ++++ REP2033 Poly TG PS + − ++++ ++++ REP2055 Poly AC PS + −++++ ++++ REP2056 Poly TC PS + − ++++ ++++ REP2057 Poly AG PS ++ − ++++++++PII = phosphodiesterase II,S1 = S1 nuclease,Exo1 = Exonuclease 1,PS = all linkages phosphorothioated,2′OMe = all riboses are 2′O methylated.− = complete degredation,++++ = no degredation,.PS = phosphorothioate,2′OMe = 2′-O-methyl modification of the ribose.These results demonstrate that phosphorothioated ONs containing onlypyrimidine nucleotides, including cytosine and/or thymidine and/or otherpyrimidines are resistant to low pH and phosphorothioated ONs containingonly cytosine nucleotides exhibit superior nuclease resistance, twoimportant characteristics for oral administration of an antiviral ON.Thus, high pyrimidine nucleotide content of an antiviral ON isadvantageous to provide resistance to low pH resistance and highcytosine content is advantagaeous to provide improved nucleaseresistance. For example, in certain embodiments, the pyrimidine contentof such an oligonucleotide is more than 50%, more than 60%, or more than70%, or more than 80%, or more than 90%, or 100%. Furthermore, theseresults show the potential of a method of treatment using oraladministration of a therapeutically effective amount of at least onepharmacologically acceptable ON composed of pyrimidine nucleotides.These results also show the potential of ONs containing high levels ofpyrimidine nucleotides as a component of an antiviral ON formulation.

Example 17 Sequence Independent Broad Spectrum Activity of ONs In Vivo

We show here that a 40 base sequence-independent PS-ON randomer haspotent antiviral activity in six different animal models of viralinfection (see table 16). The 40 base PS-ON randomer was introduced toanimals by multiple routes of administration including subcutaneous,intraperitoneal and aerosol (inhalation). These data strongly supportthe therapeutic potential of sequence independent ONs as broad spectrumantivirals. TABLE 16 PS-ON randomers have potent broad spectrum in vivoantiviral activity Virus (strain) Reduction in viral titer (organ)(Animal/mode of infection) relative to placebo (route) p-value EbolaZaire (Mayinga) 100% survival (n = 6) ND (mouse/IP, lethal model)(intraperitoneal) Influenza A/HK/68 3.8 log₁₀ (lung) <0.001 (Mouse/IN)(aerosol) MCMV (Smith) 2.67 log₁₀ (spleen) <0.0001 (Mouse/IV)(subcutaneous) 1.67 log₁₀ (spleen) 0.012 (intraperitoneal) HSV-2 ˜70% ofanimals protected from ND (Mouse/vaginal gel) HSV-2 transmissionFriend's Leukemia Virus 68%(Reduction of infected 0.0084 (Mouse/IV)splenocytes) (subcutaneous) Respiratory Syncytial Virus 1.1 log₁₀ (lung)<0.01 (Long) (Cotton rat/IN) (aerosol)ND = not determined

Example 18 Oligonucleotides have Antiviral Activity in a Broad Spectrumof Viruses

We show here that a 40 PS-ON randomer has antiviral activity in vitroagainst 13 viral families (see table 17). TABLE 17 PS-ON randomers havebroad spectrum in vitro antiviral activity Activity Family Virus(Strain) (IC₅₀, μM) Assay Used Herpesviridae HSV-1 (KOS) 0.06 Plaquereduction HSV-1 0.2 Inhibition of CPE HSV-2 (MS2) 0.1 Plaque reductionHSV-2 0.02 Inhibition of CPE CMV (AD169) 0.13 Plaque reduction Human CMV0.02 Inhibition of CPE VZV <0.02 Inhibition of CPE Retroviridae HIV-1˜0.1 p24 ELISA (multiple clinical isolates) (in human PBMCs) HIV-1(NL4-3) 0.011 Inhibition of CPE HIV/MLV Chimera 0.014 fluorescence-basedinfection assay Hepadnaviridae HBV 0.007 detection of virions in thesupernatant Paramyxoviridae RSV (A2) 0.019 inhibition of CPEParainfluenza-3 0.125 plaque reduction Coronaviridae SARS (Toronto-2)100 Inhibition of CPE Filoviridae Ebola Zaire (Mayinga) 0.1 FACSanalysis of infected cells Marburg (Muskoke) IC99 < 1 fluorescent plaquereduction Arenaviridae Lassa Fever (Josiah) IC99 < 1 reduction of virusin supernatant Bunyaviridae Hantavirus (Prospect Hill)  IC99 < 10fluorescent plaque reduction Orthopoxviridae vaccinia (WR) ˜1.5 plaquereduction vaccinia ˜0.5 plaque reduction ectromelia (mousepox) ˜1.5plaque reduction Flaviviridae West Nile 3.02 inhibition of CPE (NY-99)Yellow Fever 3.47 inhibition of CPE Dengue ˜10 plaque reduction (Den-4)Togaviridae Western Equine Encephalitis 0.12 inhibition of CPERhabdoviridae Rabies (ERA) IC99 < 1 fluorescent plaque reductionOrthomyxoviridae Influenza A ˜1 plaque reduction

Example 19 In Vivo and In Vitro Anti-Influenza Activity of PS-ONs

In order to further assess the anti-influenza activity of ONs, REP 2006was tested against different strains of influenza using ahemagluttination assay. REP 2006 displayed a broad spectrumanti-influenza activity as shown in Table 18. TABLE 18 Broad spectrumantiviral activity of a REP 2006 against multiple strains of influenza.Trial 1 IC₅₀ Trial 2 IC₅₀ Influenza strain (mM) (mM) A/New Caledonia(H1N1) 0.014 0.055 A/Taiwan (H1N1) 0.014 0.055 B/Panama 0.038 0.055B/Singapore 0.038 0.055 A/PR8 (H1N1) 0.055 0.015 A/HK/68 (H3N2) 0.0080.0017 A/WSN (H1N1) 0.038 Not tested

In order to asses the potential of ONs as drugs for the treatment ofinfluenza, REP 2006 was tested in a mouse model of influenza infection.REP 2006 was prepared at two concentrations in water for injection andaerosolized by nebulization where the outlet was connected to anAnderson cascade chamber. 20 g Balb/c mice were exposed daily toaerosolized randomer 1 for 30 minutes using 10 ml of REP 2006 at variousconcentrations in an aerosol chamber. Mice were intranasally infectedwith ˜100TCID of influenza A (H3N2, A/Hong Kong/68) and after 4 days ofinfection, animals were sacrificed and lung viral titers were determinedby hemagluttination assay. REP 2006 demonstrated a potent anti-influenzaactivity in vivo as shown in Table 19. TABLE 19 In vivo efficacy of theREP2006 against influenza A. Dose Viral titer ANOVA mg/ml - SDA (log10/glung) Non- Treatment mg/kg - IP/SC Regimen (days) Mean St. dev.Parametric parametric Influenza A/HK/68 (n = 5) in Balb/c mice dH₂O 0(SDA) −1, 0, 1, 2 6.6 0.9 NA NA ribavirin 180 (IP) −1, 0, 1, 2 3.3 0.6<0.001 NS REP 2006 10 (SDA) −1, 0, 1, 2 2.8 0.9 <0.001 NS REP 2006 100(SDA) −1, 0, 1, 2 <2.3 0.0 <0.001 <0.01 Influenza A/HK/68 (n = 5) inBalb/c mice dH₂O 0 (SDA) −1, 0, 1, 2 5.8 0.4 NA NA REP 2006 10 (SDA) −1,0, 1, 2 3.9 0.4 <0.001 NS 2 × 10 (SDA)** −1, 0, 1, 2 3.3 0.5 <0.001<0.01 2 × 100 (SDA)** 1, 2 1.1 1.5 <0.001 NS 20 (IP) 1, 2 3.2 0.4 <0.001NS 20 (SC) 1, 2 3.9 0.2 <0.001 NSSDA = small droplet aerosol,IP = intraperitoneal,SC = subcutaneous)**indicates two daily doses given 12 hours apart.

Example 20 Phosphorothioated Polypyrimidine ON Exhibits ImprovedAntiviral Activity in Acidic Environment In Vivo

In order to assess the resistance of polypyrimidine ONs to low pH andtheir capacity to be active drugs at lower pH in vivo, REP 2031 (PSpolyC) was tested in a HSV-2 vaginal mouse model. Groups of Female SwissWebster were administered a 0.1 ml suspension containing 3 mg ofmedroxyprogesterone acetate by subcutaneous injection 7 and 1 days priorto viral challenge, to increase susceptibility to vaginal HSV-2infection. The vaginal vault was swabbed twice, first with a moistenedtype 1 calcium alginate-tipped swab and then with a dry swab. Animalswere treated with 15 μl of either the candidate solution or a placebocontrol using a positive displacement pipetter. Five minutes later,animals were inoculated by instillation of 15 μl of a suspensioncontaining 10⁴ pfu of HSV-2, strain 186. Vaginal swabs samples werecollected from all animals on day 2 after inoculation and stored frozen(−80° C.) until assayed for the presence of virus by culture. Mice wereevaluated daily up to day 21 after inoculation, for evidence ofsymptomatic infection that can include hair loss and erythema around theperineum, chronic urinary incontinence, hind-limb paralysis, andmortality. Animals that did not develop symptoms were defined asinfected if virus was isolated from vaginal swab samples collected onday 2 after inoculation. Results showed (Table 20) that polypyrimidineREP 2031 had an antiviral activity in an acidic environment, such as thevagina in this example or the stomach. TABLE 20 Vaginal efficacy of ONsagainst HSV-2 Virus (strain) Route of Reduction in viral (Animal/administration titre (organ) mode of and relative to infection) Compounddosing regimen placebo (log₁₀) HSV-2 (186) REP 2006 Single prophylactic8/12 animals (Swiss Webster (PS-randomer) topical application protectedfrom mice/vaginal) to vagina transmission (100 mg/ml gel) (0/12 inuntreated animals) REP 2031 12/12 animals (PS-poly C) protected fromtransmission

All patents and other references cited in the specification areindicative of the level of skill of those skilled in the art to whichthe invention pertains, and are incorporated by reference in theirentireties, including any tables and figures, to the same extent as ifeach reference had been incorporated by reference in its entiretyindividually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, variances, andcompositions described herein as presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art, which are encompassed within the spirit of theinvention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Forexample, variations can be made to synthesis conditions and compositionsof the oligonucleotides. Thus, such additional embodiments are withinthe scope of the present invention and the following claims.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Also, unless indicated to the contrary, where various numberical valuesare provided for embodiments, additional embodiments are described bytaking any 2 different values as the endpoints of a range. Such rangesare also within the scope of the described invention.

Thus, additional embodiments are within the scope of the invention andwithin the following claims.

1. An oligonucleotide, having at least 50% of its nucleotides in saidoligonucleotide modified at the 2′-position of the ribose moiety andhaving at least 50% of its internucleotidic linkages modified, whereinsaid oligonucleotide has an antiviral activity against a target virus,said activity operating predominantly by a sequence independent mode ofaction.
 2. The oligonucleotide according to claim 1, wherein saidoligonucleotide has at least 50% of its nucleotides in saidoligonucleotide modified at the 2′-position of the ribose moiety and hasat least 80% of its internucleotidic linkages modified, wherein saidoligonucleotide has an antiviral activity against a target virus, saidactivity operating predominantly by a sequence independent mode ofaction.
 3. The oligonucleotide according to claim 1, wherein saidoligonucleotide has at least 80% of its nucleotides in saidoligonucleotide modified at the 2′-position of the ribose moiety and hasat least 80% of its internucleotidic linkages modified, wherein saidoligonucleotide has an antiviral activity against a target virus, saidactivity operating predominantly by a sequence independent mode ofaction.
 4. The oligonucleotide according to claim 1, wherein saidoligonucleotide has at least 90% of its nucleotides in saidoligonucleotide modified at the 2′-position of the ribose moiety andhaving at least 90% of its internucleotidic linkages modified, whereinsaid oligonucleotide has an antiviral activity against a target virus,said activity operating predominantly by a sequence independent mode ofaction.
 5. The oligonucleotide according to claim 1, wherein saidoligonucleotide has at least 100% of its nucleotides in saidoligonucleotide modified at the 2′-position of the ribose moiety andhaving at least 100% of its internucleotidic linkages modified, whereinsaid oligonucleotide has an antiviral activity against a target virus,said activity operating predominantly by a sequence independent mode ofaction.
 6. The oligonucleotide of claim 1, wherein the modified linkagesare selected from the group consisting of phosphorothioate linkages,phosphorodithioate linkages, and boranophosphate linkages.
 7. Theoligonucleotide of claim 5, wherein the modified linkages are selectedfrom the group consisting of phosphorothioate linkages,phosphorodithioate linkages, and boranophosphate linkages.
 8. Theoligonucleotide of claim 1, wherein at least 50% of the nucleotides insaid oligonucleotide comprises 2′-OMe moieties at the 2′-position of theribose moiety.
 9. The oligonucleotide of claim 5, wherein at least 100%of the nucleotides in said oligonucleotide comprises 2′-OMe moieties atthe 2′-position of the ribose moiety.
 10. The oligonucleotide of claim1, wherein at least 50% of the nucleotides in said oligonucleotidecomprise 2′-methoxyethoxy substitutions at the 2′-position of the ribosemoiety.
 11. The oligonucleotide of claim 5, wherein at least 100% of thenucleotides in said oligonucleotide comprise 2′-methoxyethoxysubstitutions at the 2′-position of the ribose moiety.
 12. Theoligonucleotide of claim 1, wherein said oligonucleotide is at least 30nucleotides in length.
 13. The oligonucleotide of claim 1, wherein saidoligonucleotide is at least 40 nucleotides in length.
 14. Theoligonucleotide of claim 1, comprising a homopolymer sequence of atleast 10 contiguous nucleotides selected from the group consisting of A,T, U, C, G, and I.
 15. The oligonucleotide of claim 1, comprising asequence of at least 10 nucleotides in length selected from the groupconsisting of polyAT, polyAC, polyAG, polyAU, polyAI, polyGC, polyGT,polyGU, polyGI, polyCT, polyCU, polyCI, and polyTI.
 16. Theoligonucleotide of claim 1, wherein at least 15% of the nucleotides insaid oligonucleotide comprise 2′-methoxyethoxy or 2′OMe substitutions atthe 2′-position of the ribose moiety.
 17. The oligonucleotide of claim1, wherein said oligonucleotide is a concatemer consisting of two ormore oligonucleotide sequences joined by a linker.
 18. Theoligonucleotide of claim 1, wherein said oligonucleotide is linked orconjugated at one or more nucleotide residues, to a molecule modifyingthe characteristics of the oligonucleotide to obtain one or morecharacteristics selected from the group consisting of higher stability,lower serum interaction, higher cellular uptake, higher viral proteininteraction, an improved ability to be formulated for delivery, adetectable signal, higher antiviral activity, better pharmacokineticproperties, specific tissue distribution, lower toxicity.
 19. Theoligonucleotide of claim 1, wherein said oligonucleotide is doublestranded.
 20. The oligonucleotide of claim 1, wherein saidoligonucleotide targets a DNA virus or a RNA virus.
 21. Theoligonucleotide of claim 1, wherein said oligonucleotide targets amember of the group consisting of herpesviridae, HSV-1, HSV-2, CMVVaricella Zoster Virus, Epstein Barr Virus, Human Herpesvirus 6A and 6B,hepadnaviridae, HBV, parvoviridae, poxviridae, papillomaviridae,adenoviridae, retroviridae, HIV-1, HIV-2, paramyxoviridae, RSV,parainfluenza virus, human metapneumovirus, bunyaviridae, hantavirus,Rift Valley fever virus, Crimean Congo Hemorrhagic Fever virus,picornaviridae, coxsackievirus, rhinovirus, flaviviridae, yellow fevervirus, dengue virus, West Nile virus, hepatitis C virus, filoviridae,Ebola virus, Marburg virus, orthomyxoviridae, influenza virus,togaviridae, Western Equine Encephalitis virus, coronaviridae,reoviridae rhabdoviridae, arenaviridae, lassa fever virus andcalciviridae.
 22. An oligonucleotide as set forth in any one of REP1001, REP 2001, REP 3007, REP 2004, REP 2005, REP 2006, REP 2007, REP2008, REP 2017, REP 2018, REP 2020, REP 2021, REP 2024, REP 2036, A20,G20, C20, REP 2029, REP 2031, REP 2030, REP 2033, REP 2055, REP 2056,REP 2057, REP 2060 and REP
 2107. 23. An oligonucleotide mixturecomprising a mixture of at least two different antiviraloligonucleotides of claim
 1. 24. An oligonucleotide mixture comprising amixture of at least ten different antiviral oligonucleotides of claim 1.25. An antiviral pharmaceutical composition comprising a therapeuticallyeffective amount of at least one pharmacologically acceptable, antiviraloligonucleotide as defined in claim 1; and a pharmaceutically acceptablecarrier.
 26. A kit comprising at least one antiviral oligonucleotide asdefined in claim 1, in a labeled package, wherein the antiviral activityof said oligonucleotide occurs principally by a non-sequencecomplementary mode of action and the label on said package indicatesthat said antiviral oligonucleotide can be used against at least onevirus.
 27. The kit of claim 26, wherein said kit contains a mixture ofat least two different antiviral oligonucleotides.
 28. A method for theprophylaxis or treatment of a viral infection in a subject, comprisingadministering to a subject in need of such a treatment a therapeuticallyeffective amount of at least one pharmacologically acceptableoligonucleotide as defined in claim
 1. 29. A method for the prophylaxisor treatment of a viral infection in a subject, comprising administeringto a subject in need of such a treatment a therapeutically effectiveamount of at least one pharmacologically acceptable oligonucleotidemixture as defined in claim
 23. 30. A method for the prophylaxis ortreatment of a viral infection in a subject, comprising administering toa subject in need of such a treatment a therapeutically effective amountof at least one pharmacologically acceptable antiviral pharmaceuticalcomposition as defined in claim
 25. 31. A method for the prophylactictreatment of cancer caused by oncoviruses in a human or a non-humananimal, comprising administering to a subject in need of such atreatment a therapeutically effective amount of at least onepharmacologically acceptable oligonucleotide as defined in claim
 1. 32.A method for the prophylactic treatment of cancer caused by oncovirusesin a human or a non-human animal, comprising administering to a subjectin need of such a treatment a therapeutically effective amount of atleast one pharmacologically acceptable oligonucleotide mixture asdefined in claim
 23. 33. A method for the prophylactic treatment ofcancer caused by oncoviruses in a human or a non-human animal,comprising administering to a subject in need of such a treatment atherapeutically effective amount of at least one pharmacologicallyacceptable antiviral pharmaceutical composition as defined in claim 25.34. An antiviral pharmaceutical composition comprising a therapeuticallyeffective amount of at least one pharmacologically acceptable,polypyrimidine oligonucleotide and a pharmaceutically acceptablecarrier, wherein the antiviral activity of said oligonucleotide occursprincipally by a sequence independent mode of action.
 35. The antiviralpharmaceutical composition of claim 34, wherein the oligonucleotidecomprises at least one modified internucleotidic linkage.
 36. Thecomposition of claim 34, wherein said composition is formulated foradministration to an acidic in vivo site.
 37. The composition of claim34, wherein said composition is adapted for oral, vaginal, or topicaladministration.
 38. The composition of claim 34, wherein saidcomposition comprises at least one polyC oligonucleotide.
 39. Thecomposition of claim 34, wherein said composition comprises at least onepolyT oligonucleotide.
 40. The composition of claim 34, wherein saidcomposition comprises at least one polyCT oligonucleotide.
 41. A methodfor the prophylaxis or treatment of a viral infection in an acidicenvironnement in a subject, comprising administering to a subject inneed of such a treatment a therapeutically effective amount of at leastone pharmacologically acceptable antiviral pharmaceutical composition asdefined in claim 34, said composition being adapted for administrationto an acidic in vivo site.
 42. An oligonucleotide, having at least 50%of its internucleotidic linkages modified, wherein said oligonucleotidehas an antiviral activity against a target virus, said activityoperating predominantly by a sequence independent mode of action, saidoligonucleotide comprising at least 80% of pyrimidine residues.
 43. Theoligonucleotide of claim 42, wherein said oligonucleotide has at least80% of its internucleotidic linkages modified.
 44. The oligonucleotideof claim 42, wherein said oligonucleotide has at least 80% of itsinternucleotidic linkages modified and has 100% of pyrimidine residues.45. The oligonucleotide of claim 42, wherein said oligonucleotide has100% of its internucleotidic linkages modified and has at least 80% ofpyrimidine residues.
 46. The oligonucleotide of claim 42, wherein saidoligonucleotide has 100% of its internucleotidic linkages modified andhas 100% of pyrimidine residues.
 47. The oligonucleotide of claim 42,wherein the modified linkages are selected from the group consisting ofphosphorothioate linkages, phosphorodithioate linkages, andboranophosphate linkages.
 48. The oligonucleotide of claim 46, whereinthe modified linkages are selected from the group consisting ofphosphorothioate linkages, phosphorodithioate linkages, andboranophosphate linkages.
 49. The oligonucleotide of claim 42, whereinthe modified linkages are phosphorothioate linkages
 50. Theoligonucleotide of claim 48, wherein the modified linkages arephosphorothioate linkages.
 51. The oligonucleotide of claim 42, whereinthe pyrimidine residues are cytosine residues.
 52. The oligonucleotideof claim 46, wherein the pyrimidine residues are cytosine residues. 53.The oligonucleotide of claim 42, wherein the pyrimidine residues arethymine residues.
 54. The oligonucleotide of claim 46, wherein thepyrimidine residues are thymine residues.
 55. The oligonucleotide ofclaim 42, wherein the pyrimidine residues are cytosine or thymineresidues.
 56. The oligonucleotide of claim 46, wherein the pyrimidineresidues are cytosine or thymine residues.
 57. The oligonucleotide ofclaim 42, wherein said oligonucleotide is at least 30 nucleotides inlength.
 58. The oligonucleotide of claim 46, wherein saidoligonucleotide is at least 30 nucleotides in length.
 59. Theoligonucleotide of claim 42, wherein said oligonucleotide is at least 40nucleotides in length.
 60. The oligonucleotide of claim 46, wherein saidoligonucleotide is at least 40 nucleotides in length.
 61. Theoligonucleotide of claim 42, wherein at least 15% of the nucleotides insaid oligonucleotide comprise 2′-methoxyethoxy or 2′-OMe substitutionsat the 2′-position of the ribose moiety.
 62. The oligonucleotide ofclaim 42, wherein said oligonucleotide is a concatemer consisting of twoor more oligonucleotide sequences joined by a linker.
 63. Theoligonucleotide of claim 46, wherein said oligonucleotide is aconcatemer consisting of two or more oligonucleotide sequences joined bya linker.
 64. The oligonucleotide of claim 42, wherein saidoligonucleotide is linked or conjugated at one or more nucleotideresidues, to a molecule modifying the characteristics of theoligonucleotide to obtain one or more characteristics selected from thegroup consisting of higher stability, lower serum interaction, highercellular uptake, higher viral protein interaction, an improved abilityto be formulated for delivery, a detectable signal, higher antiviralactivity, better pharmacokinetic properties, specific tissuedistribution, lower toxicity.
 65. The oligonucleotide of claim 46,wherein said oligonucleotide is linked or conjugated at one or morenucleotide residues, to a molecule modifying the characteristics of theoligonucleotide to obtain one or more characteristics selected from thegroup consisting of higher stability, lower serum interaction, highercellular uptake, higher viral protein interaction, an improved abilityto be formulated for delivery, a detectable signal, higher antiviralactivity, better pharmacokinetic properties, specific tissuedistribution, lower toxicity.
 66. The oligonucleotide of claim 42,wherein said oligonucleotide is double stranded.
 67. The oligonucleotideof claim 46, wherein said oligonucleotide is double stranded.
 68. Theoligonucleotide of claim 42, wherein said oligonucleotide targets a DNAvirus or a RNA virus.
 69. The oligonucleotide of claim 46, wherein saidoligonucleotide targets a DNA virus or a RNA virus.
 70. Theoligonucleotide of claim 42, wherein said oligonucleotide targets amember of the group consisting of herpesviridae, HSV-1, HSV-2, CMVVaricella Zoster Virus, Epstein Barr Virus, Human Herpesvirus 6A and 6B,hepadnaviridae, HBV, parvoviridae, poxviridae, papillomaviridae,adenoviridae, retroviridae, HIV-1, HIV-2, paramyxoviridae, RSV,parainfluenza virus, human metapneumovirus, bunyaviridae, hantavirus,Rift Valley fever virus, Crimean Congo Hemorrhagic Fever virus,picornaviridae, coxsackievirus, rhinovirus, flaviviridae, yellow fevervirus, dengue virus, West Nile virus, hepatitis C virus, filoviridae,Ebola virus, Marburg virus, orthomyxoviridae, influenza virus,togaviridae, Western Equine Encephalitis virus, coronaviridae,reoviridae rhabdoviridae, arenaviridae, lassa fever virus andcalciviridae.
 71. The oligonucleotide of claim 46, wherein saidoligonucleotide targets a member of the group consisting ofherpesviridae, HSV-1, HSV-2, CMV Varicella Zoster Virus, Epstein BarrVirus, Human Herpesvirus 6A and 6B, hepadnaviridae, HBV, parvoviridae,poxviridae, papillomaviridae, adenoviridae, retroviridae, HIV-1, HIV-2,paramyxoviridae, RSV, parainfluenza virus, human metapneumovirus,bunyaviridae, hantavirus, Rift Valley fever virus, Crimean CongoHemorrhagic Fever virus, picornaviridae, coxsackievirus, rhinovirus,flaviviridae, yellow fever virus, dengue virus, West Nile virus,hepatitis C virus, filoviridae, Ebola virus, Marburg virus,orthomyxoviridae, influenza virus, togaviridae, Western EquineEncephalitis virus, coronaviridae, reoviridae rhabdoviridae,arenaviridae, lassa fever virus and calciviridae.
 72. An oligonucleotidemixture comprising a mixture of at least two different antiviraloligonucleotides of claim
 42. 73. An oligonucleotide mixture comprisinga mixture of at least two different antiviral oligonucleotides of claim46.
 74. An oligonucleotide mixture comprising a mixture of at least tendifferent antiviral oligonucleotides of claim
 42. 75. An oligonucleotidemixture comprising a mixture of at least ten different antiviraloligonucleotides of claim
 46. 76. An antiviral pharmaceuticalcomposition comprising a therapeutically effective amount of at leastone pharmacologically acceptable, antiviral oligonucleotide as definedin claim 42; and a pharmaceutically acceptable carrier.
 77. An antiviralpharmaceutical composition comprising a therapeutically effective amountof at least one pharmacologically acceptable, antiviral oligonucleotideas defined in claim 46; and a pharmaceutically acceptable carrier. 78.The composition of claim 76, wherein said composition is formulated foradministration to an acidic in vivo site.
 79. The composition of claim77, wherein said composition is formulated for administration to anacidic in vivo site.
 80. The composition of claim 76, wherein saidcomposition is adapted for oral, vaginal, or topical administration. 81.The composition of claim 77, wherein said composition is adapted fororal, vaginal, or topical administration.
 82. A kit comprising at leastone antiviral oligonucleotide as defined in claim 42, in a labeledpackage, wherein the antiviral activity of said oligonucleotide occursprincipally by a non-sequence complementary mode of action and the labelon said package indicates that said antiviral oligonucleotide can beused against at least one virus.
 83. A kit comprising at least oneantiviral oligonucleotide as defined in claim 46, in a labeled package,wherein the antiviral activity of said oligonucleotide occursprincipally by a non-sequence complementary mode of action and the labelon said package indicates that said antiviral oligonucleotide can beused against at least one virus.
 84. The kit of claim 78, wherein saidkit contains a mixture of at least two different antiviraloligonucleotides.
 85. The kit of claim 83, wherein said kit contains amixture of at least two different antiviral oligonucleotides.
 86. Amethod for the prophylaxis or treatment of a viral infection in asubject, comprising administering to a subject in need of such atreatment a therapeutically effective amount of at least onepharmacologically acceptable oligonucleotide as defined in claim
 42. 87.A method for the prophylaxis or treatment of a viral infection in asubject, comprising administering to a subject in need of such atreatment a therapeutically effective amount of at least onepharmacologically acceptable oligonucleotide as defined in claim
 46. 88.A method for the prophylaxis or treatment of a viral infection in asubject, comprising administering to a subject in need of such atreatment a therapeutically effective amount of at least onepharmacologically acceptable oligonucleotide mixture as defined in claim72.
 89. A method for the prophylaxis or treatment of a viral infectionin a subject, comprising administering to a subject in need of such atreatment a therapeutically effective amount of at least onepharmacologically acceptable oligonucleotide mixture as defined in claim73.
 90. A method for the prophylaxis or treatment of a viral infectionin a subject, comprising administering to a subject in need of such atreatment a therapeutically effective amount of at least onepharmacologically acceptable antiviral pharmaceutical composition asdefined in claim
 76. 91. A method for the prophylaxis or treatment of aviral infection in a subject, comprising administering to a subject inneed of such a treatment a therapeutically effective amount of at leastone pharmacologically acceptable antiviral pharmaceutical composition asdefined in claim
 77. 92. A method for the prophylactic treatment ofcancer caused by oncoviruses in a human or a non-human animal,comprising administering to a subject in need of such a treatment atherapeutically effective amount of at least one pharmacologicallyacceptable oligonucleotide as defined in claim
 42. 93. A method for theprophylactic treatment of cancer caused by oncoviruses in a human or anon-human animal, comprising administering to a subject in need of sucha treatment a therapeutically effective amount of at least onepharmacologically acceptable oligonucleotide as defined in claim
 46. 94.A method for the prophylactic treatment of cancer caused by oncovirusesin a human or a non-human animal, comprising administering to a subjectin need of such a treatment a therapeutically effective amount of atleast one pharmacologically acceptable oligonucleotide mixture asdefined in claim
 72. 95. A method for the prophylactic treatment ofcancer caused by oncoviruses in a human or a non-human animal,comprising administering to a subject in need of such a treatment atherapeutically effective amount of at least one pharmacologicallyacceptable oligonucleotide mixture as defined in claim
 73. 96. A methodfor the prophylactic treatment of cancer caused by oncoviruses in ahuman or a non-human animal, comprising administering to a subject inneed of such a treatment a therapeutically effective amount of at leastone pharmacologically acceptable antiviral pharmaceutical composition asdefined in claim
 76. 97. A method for the prophylactic treatment ofcancer caused by oncoviruses in a human or a non-human animal,comprising administering to a subject in need of such a treatment atherapeutically effective amount of at least one pharmacologicallyacceptable antiviral pharmaceutical composition as defined in claim 77.98. An antiviral pharmaceutical composition comprising a therapeuticallyeffective amount of at least one pharmacologically acceptable,polypyrimidine oligonucleotide and a pharmaceutically acceptablecarrier, wherein the antiviral activity of said oligonucleotide occursprincipally by a sequence independent mode of action; and apharmaceutically acceptable carrier.
 99. The antiviral pharmaceuticalcomposition of claim 98, wherein the oligonucleotide comprises modifiedinternucleotidic linkages.
 100. The composition of claim 98, whereinsaid composition is formulated for administration to an acidic in vivosite.
 101. The composition of claim 98, wherein said composition is inthe form of a powder.
 102. The composition of claim 98, wherein saidcomposition is adapted for oral, vaginal, or topical administration.103. The composition of claim 98, wherein said composition comprises atleast one polyC oligonucleotide.
 104. The composition of claim 98,wherein said composition comprises at least one polyT oligonucleotide.105. The composition of claim 98, wherein said composition comprises atleast one polyCT oligonucleotide.
 106. A method for the prophylaxis ortreatment of a viral infection in an acidic environnement in a subject,comprising administering to a subject in need of such a treatment atherapeutically effective amount of at least one pharmacologicallyacceptable antiviral pharmaceutical composition as defined in claim 42,said composition being adapted for administration to an acidic in vivosite.
 107. A method for the prophylaxis or treatment of a viralinfection in an acidic environnement in a subject, comprisingadministering to a subject in need of such a treatment a therapeuticallyeffective amount of at least one pharmacologically acceptable antiviralpharmaceutical composition as defined in claim 46, said compositionbeing adapted for administration to an acidic in vivo site.